The following are frequently asked questions about our products and services. If you have a question that is not answered on this page, or if you want clarification, please feel free to contact us. Our customer service and technical support teams are always glad to help.
Call Toll free in the US 888-810-6168 or 858-642-2058 or email us at tech@ebioscience.com.
GENERAL INFORMATION:
FLOW CYTOMETRY:
IMMUNOASSAY:
BIOASSAY:
WESTERN BLOT (WB)/IMMUNOPERCIPITATION (IP):
IMMUNOHISTOCHEMISTRY:
Q: What type of antibodies do you offer?
eBioscience® offers a wide range of monoclonal and polyclonal antibodies for detecting murine, human, non-human primate and other species of antigens. Antibodies can be used for the detection of cell surface markers, such as CD molecules, cytoskeletal and cytoplasmic proteins, chemokine receptors, as well as intracellular cytokines/chemokines and intra-nuclear proteins including transcription factors.
Q: What is the shelf life of your antibodies?
The majority of eBioscience antibodies have a guaranteed shelf life of one year from date of receipt unless indicated on our Technical Data Sheets. This guarantee is provided if the products are kept under optimal storage conditions as stated on the Technical Data Sheet. If you are not completely satisfied with the performance of your product, please contact technical support at tech@ebioscience.com for assistance.
Q: What information do you have regarding the cross-reactivity of your antibodies?
Information about known cross-reactivity of an antibody is given on the Technical Data Sheets. We have done some in-house testing to analyze cross-reactivity of anti-human antibodies amongst non-human primates. The results of these tests are summarized in our Cross-Reactivity Charts.
Q: What formats do you offer?
For convenience, eBioscience offers antibodies in a variety of formats, Best Protocols® Fluorescent Dyes Chart. We carry purified and biotin conjugates with and without sodium azide and a large number of direct fluorochrome conjugates. Our newest additions are eFluor Nanocrystals (NC) and eFluor Organic dyes, for mor information please click here. These include eFluor 605NC, 625NC, 650NC (all nanocrystal reagents) as well as eFluor 450 (Pacific Blue replacement), APC-eFluor 780 (APC-Alexa® Fluor 750 replacement), eFluor 660 (Alexa Fluor 647 replacement) and PerCP-eFluor 710 .
Q: What is the difference between your Affinity Purified and Functional Grade Purified formats?
The Affinity Purified format of our monoclonal antibodies contains no additional protein carriers such as BSA or gelatin but does contain sodium azide as a preservative. These products can be used for flow cytometry, WB, IP, and IHC applications. The Functional Grade purified formats contain no sodium azide and are tested for endotoxin. These products meet or exceed the industry standard of 0.01ng/ug endotoxin/ug antibody and in most instances have endotoxin levels significantly lower than these standards. Please contact tech@ebioscience.com if you would like lot specific information for Functional Grade products. The Functional Grade formats are generally used for bioassays and in vivo studies.
Q: What are the concentrations of your antibodies?
eBioscience offers both test and microgram to milligram sizes, depending on the product. The concentration of the test sizes is indicated on the Technical Data Sheets under Applications Tested. The concentrations of the microgram sizes vary depending on the format. Please refer to the product vial for lot specific information.
Q: What applications can I use eBioscience antibodies for?
eBioscience antibodies can be used for a variety of applications, such as:
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Flow cytometric analysis- surface antigen staining (FC)
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Flow cytometric analysis- intracellular staining (IC Flow)
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Immunohistochemistry (IHC)
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Immunohistochemistry- frozen sections (IH/F)
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Immunohistochemistry- paraffin sections (IHC-Paraffin)
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Immunoblotting (WB)
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Immunoprecipitation (IP)
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ELISA
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ELISPOT
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Bioassays (FA)
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Neutralization/Blocking studies (NU)
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In vitro Cytokine Capture Assay (IVCCA)
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In vivo studies in mice
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Chromatin immunoprecipitation (ChIP)
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Q: Which biomolecules can be conjugated to eFluor® Nanocrystals?
eFluor® Nanocrystals have been successfully conjugated to peptides, proteins and oligonucleotides.
Q: Which protocol do I use for conjugation of a biomolecule to eFluor® Nanocrystals?
Protocols for conjugation of either amine or carboxyl eFluor® Nanocrystals are provided in our Best Protocols®. These protocols will also be provided with your order of Reactive eFluor® Nanocrystals.
Q: Can I conjugate eFluor Nanocrystals to an antibody myself?
Reactive eFluor Nanocrystals, in a variety of emission spectra, with both carboxyl and amine surface chemistries are available for conjugation. Conjugation protocols are available in our Best Protocols® and are provided when you order Reactive eFluor Nanocrystals. For more details, please see our Tools for Discovery product listing.
Q: Which kit should I choose: ‘Amine-Reactive’ or ‘Sulfhydryl-Reactive’?
The most important factor in choosing the kit is knowing which functional group is available on your biomolecule. If your molecule has a primary amine, then you should choose the ‘Amine-Reactive’ Kit. If your molecule has a sulfhydryl group (thiol, disulfide, or cysteine residue), then you would choose the ‘Sulfhydryl-Reactive’ Kit. Antibodies, for example, have both functional groups available, and you can choose either kit.
Q: Do both Sulfhydryl-Reactive and Amine-Reactive kits perform equally well in all applications?
We have not tested all applications. Both kits have been extensively tested in flow cytometry, immunocytochemistry (ICC) and immunohistochemistry (IHC). Although both kits give good results in these applications, we have found that the Sulfhydryl-Reactive kit may demonstrate a slight advantage for flow cytometry applications, and the Amine-Reactive kit may demonstrate a slight advantage for ICC/IHC applications but this will vary depending on the particular protein. It is critical for optimal performance to use the recommended protocols and buffers as stated in our Best Protocols.
Q: How many functional groups are there on each eFluor® Nanocrystal?
There are an estimated 30–60 functional reactive groups on each eFluor® Nanocrystal.
Q: How much eFluor® Nanocrystal is included in each kit?
A: The kits are optimized for the conjugation of 200 µg of antibody per reaction at a ratio of 2:1 (for the eF605NC) and 4:1 (for the eF650NC) antibody to eFluor® Nanocrystal. Therefore, each reaction in the kit contains 0.67 nmol of reactive eFluor® Nanocrystal for the eF605NC and 0.33 nmol for the eF650NC.
Q: What types of proteins can I conjugate?
Please refer to Other Proteins Section.
Q: I have finished conjugating my biomolecule to the eFluor® Nanocrystal, can I freeze the conjugate for long-term storage.
No, freezing eFluor® Nanocrystal conjugates is detrimental to their performance. eFluor® Nanocrystal conjugates should be stored at 2-8°C in the dark. We recommend storing your vials away from the back of the refrigerator where samples may come close to freezing, which will affect performance.
Q: I followed your protocol exactly to conjugate my antibody to the eFluor® Nanocrystals, but I see no signal in my application. What should I do?
Certain antibodies may perform better using one chemistry over the other. For example, if an amine group is present in the antigen binding site, conjugation with the Amine-Reactive conjugation kit to this amine may block binding of the antibody to its epitope. Alternatively, if the disulfide linkages in your antibody are blocked or protected, the Sulfhydryl-Reactive kit will not work because conjugation to the eFluor® Nanocrystals will not occur. In either case, the best solution is to try the alternative kit chemistry. Please see the section(s) below concerning the application you are performing for additional troubleshooting assistance.
Q: Does it matter what my final conjugation volume and antibody concentration is once I add the antibody to the eFluor® Nanocrystal solution?
The eFluor® Nanocrystal Conjugation Kits are optimized for a total conjugation volume of 300 µL. However, you may achieve slightly better results if the conjugation volume is kept at approximately 100 µL total conjugation volume. Conjugation volumes as high as 550 µL have been tested, showing that the larger conjugation volume may result in a slight reduction in performance of the final conjugate.
Q: Do I need to perform the conjugation reaction in the dark?
No, there is no significant difference between eFluor® Nanocrystal conjugations done in ambient light as compared to in the dark.
Q: Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance in either the Sulfhydryl-Reactive or Amine-Reactive Kits. However, we recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates (for example, if the sulfhydryl or amine groups are sterically-hindered). Conjugation times longer than 2 hours do not increase the conjugation efficiency.
Q: Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor® Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor® Nanocrystal Conjugated protein.
Q: What is the shelf-life of my eFluor® Nanocrystal conjugate?
This may vary depending on the type of biomolecule attached, but we always recommend using the conjugate immediately for best results. However, conjugates are stable for 4-6 months after conjugation when stored properly (Stored at 2-8°C in the dark).
Antibody
Q: My biomolecule is in a buffer containing sodium azide. Will this interfere with the conjugation?
We have verified that sodium azide concentrations up to 0.09% (w/v) do not interfere with either the Sulfhydryl-Reactive or the Amine-Reactive eFluor® Nanocrystal Conjugation Kits.
Q: My biomolecule is in a high/low pH buffer, will this affect the conjugation reaction?
Yes, if the biomolecule is present in a buffer outside the pH range (7-9 for the Amine-Reactive kit and 6.5-7.5 for the Sulfhydryl-Reactive kit), you should buffer exchange it into a buffer such as PBS or Borate in the pH range from 6-8, and concentrate it into 100 – 300 µL total volume prior to use.
Q: My antibody is in a buffer containing carrier proteins such as serum or BSA. Will this interfere with the conjugation?
Yes, carrier proteins will interfere with both the Amine-Reactive and Sulfhydryl-Reactive eFluor® Nanocrystal Conjugation Kits. We suggest that you purify your antibody on a Protein G or Protein A resin and resuspend in PBS or TBS buffer.
Q: What if I want to conjugate a molecule that is not an antibody?
When conjugating new biomolecules to eFluor® Nanocrystals, it is recommended to perform a titration to verify and optimize the performance of the conjugate. Other biomolecules should conjugate to the eFluor® Nanocrystal provided that the biomolecule contains the appropriate functional group for conjugation, (free sulfhydryl or disulfide bridge for the Sulfhydryl-Reactive kit, or a primary amine for the Amine-Reactive kit) and the molecular weight is between 30-150 kDa (Amine-Reactive kit) or less than 150 kDa (Sulfhydryl-Reactive kit).
Q: My biomolecule has a molecular weight greater than 150 kDa. Will the conjugation kit work for me?
Although the conjugation will still likely occur, the purification of molecular weights greater than 150 kDa using our eFluor® Nanocrystal Kits is unlikely. You can still use the kit(s), but you will have to purify the conjugate from un-reacted biomolecules using a technique capable of resolving the two molecular weights. Effective techniques for separation include size-exclusion chromatography, ion-exchange chromatography, or hydrophobic interaction chromatography.
Q: My biomolecule has a molecular weight lower than 30 kDa. Will the conjugation kit work for me?
For the eFluor® Nanocrystal Sulfhydryl-Reactive kit, this will not be a problem. During the protocol for the Amine-Reactive kit, however, your modified biomolecule will not elute from the spin column, therefore molecules with low molecular weights cannot be used with the Amine-Reactive kit at this time.
Q: My biomolecule is in a buffer containing sodium azide. Will this interfere with the conjugation?
We have verified that sodium azide concentrations up to 0.09% (w/v) do not interfere with either the eFluor® Nanocrystal Sulfhydryl-Reactive or Amine-Reactive eFluor® Nanocrystal Conjugation Kits.
Q: My biomolecule is in a high/low pH buffer, will this affect the conjugation reaction?
Yes, if the biomolecule is present in a buffer outside the pH range of 6-8, you should buffer exchange it into a buffer such as PBS or Borate in the pH range (7-9 for the Amine-Reactive kit and 6.5-7.5 for the Sulfhydryl-Reactive kit), and concentrate it into 100 – 300 µL total volume prior to use.
Sulfhydryl-Reactive Kit Protocol Questions:
Q: What if I don’t have a 60°C water bath?
Take a 500 mL glass beaker and fill it with 250 mL H2O. Either heat the beaker in a microwave, or place it onto a hot plate until the temperature reaches ~60°C. If the temperature gets too hot, add cool tap water to adjust.
Q: My molecule has a disulfide, not a sulfhydryl functional group. Will that still conjugate using the eFluor® Nanocrystal Sulfhydryl-Reactive kit?
Yes, free sulfhydryl groups as well as disulfide bonds and cysteine groups may be used to conjugate to the Sulfhydryl-Reactive eFluor® Nanocrystal Conjugation Kit.
Q: Do I need to reduce my molecule using Β-ME or DTT prior to use?
No, you do not have to reduce your molecule. You can simply add the molecule to the reconstituted eFluor® Nanocrystals and proceed with the conjugation.
Q: How will the Sulfhydryl-Reactive kit affect the function of my protein?
The eFluor® Nanocrystal Sulfhydryl-Reactive kit will reduce any disulfides to free sulfhydryl functional groups and then form a covalent bond to the eFluor® Nanocrystal outer coating. If your protein has structural components or activity that is dependent upon the disulfide bond, then you may achieve better results with the eFluor® Nanocrystal Amine-Reactive kit.
Q: How does the Sulfhydryl-Reactive kit work?
The Sulfhydryl-Reactive kit contains an eFluor® Nanocrystal formulation containing a unique reducing agent and maleimide-functionalized nanocrystals. The reduced sulfhydryl group from the biomolecule conjugates in-situ to the eFluor® Nanocrystal on the outer coating.
Q: Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance in either the Sulfhydryl-Reactive or Amine-Reactive Kits. However, we recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates (for example, if the sulfhydryl or amine groups are sterically-hindered). Conjugation times longer than 2 hours do not increase the conjugation efficiency.
Q: Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor® Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor® Nanocrystal Conjugated protein.
Amine-Reactive Kit Protocol Questions:
Q: If I use a lower antibody concentration (0.5 mg/mL in 400 µL) for the Amine-Reactive kit, will additional steps/supplies be required?
Yes, because each spin column provided in the kit can only de-salt a volume of 100 µL, you will either need 4 spin columns (2 are provided) to de-salt the activated antibody solution (400 µL), or you will need to concentrate the antibody solution down to 100 µL and use a single spin column, as detailed in the protocol.
Q: How will the Amine-Reactive kit affect the function of my protein?
The Amine-Reactive kit will modify available primary amine groups on your protein and then conjugate to the eFluor® Nanocrystal surface through those modifications. If the active portion of your biomolecule is known to contain primary amine functionalities, conjugation of the eFluor® Nanocrystals to these amines could block activity of your biomolecule. In this instance, you may achieve better results with the Sulfhydryl-Reactive eFluor® Nanocrystal kit.
Q: How does the Amine-Reactive kit work?
The Amine-Reactive kit will first activate available primary amine functional groups on your biomolecule with a hydrazine moiety. Hydrazine is specific for a coupling reaction with our formylbenzamide-modified eFluor® Nanocrystals.
Q: Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance. However, we do recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates.
Q: Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor® Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor® Nanocrystal Conjugated protein.
Questions about Applications for eFluor® NC conjugated molecules:
Q: Do both Sulfhydryl-Reactive and Amine-Reactive kits perform equally well in all applications?
We have not tested all applications. Both kits have been optimized for flow cytometry as well as immunocytochemistry (ICC) and immunohistochemistry (IHC). Although both kits generate good results in these applications, we have found that the Sulfhydryl-Reactive kit may demonstrate a slight advantage for flow cytometry applications, and the Amine-Reactive kit may demonstrate a slight advantage for ICC/IHC applications but this will vary depending on the particular protein. It is critical for optimal performance, to use the recommended protocols and buffers as stated in our BestProtocols.
Also refer to eFluor® NC FAQs
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Q: What are good fluorochrome dyes to use in multi-color staining for flow cytometry?
For detailed information about the absorption and emission wavelengths and intensity of the fluorochrome dyes we offer, see our BestProtocols® Fluorescent Dyes Chart or our Fluorochrome Poster. We are more than happy to help you plan your experiments to match the antibodies with the best fluorochromes. Please contact tech@ebioscience.com should you require furthur information.
Q: What is compensation?
Compensation is a technique used to eliminate false signal that results from spectral overlap between fluorescent dyes when used in multicolor staining panels. For example, FITC has a broad emission spectra that spills into the PE detector. As a result, the cytometer will register a false population of PE labeled cells. Corrections must be made to avoid this. For further information on compensation visit this website www.drmr.com.
Q: What antibodies can I use to label a particular cell type for flow cytometric analysis?
Use our Mouse and Human CD charts to help identify the expression pattern of the different antigens of interest. There you will also find information about staining and links to product information regarding each antibody eBioscience has available.
Q: How do I discriminate between live cells and dead cells when doing a flow cytometry analysis?
Discrimination between viable and non-viable cells can be carried out with the use of the 7-AAD viability dye (cat. 00-6993) or Propidium Iodide (PI) Staining Solution (cat. 00-6990). The 7-AAD or PI will mark the non-viable cells by binding to the nuclei of those cells. The nucleic acid of viable cells will not be accessible to the dye and will not be stained. When analyzing the data collected, gate out all cells stained with the viability dye. When staining for intracellular proteins, use the Fixable Viability Dye eFluor 450, 660, or 780. For more information, please see our Functional Dyes and Reagents webpage.
Q:What do Monensin and Brefeldin A do?
These are critical chemicals for inclusion in cell cultures during cell activation prior to analysis by flow cytometry for the presence of intracellular chemokines and cytokines. Monensin (cat. 00-4505) and Brefeldin A (cat. 00-4506) are protein transport inhibitors that block secretion of proteins by cells via the golgi apparatus, thereby causing an accumulation of cytokines at the endoplasmic reticulum or Golgi. Cells are often incubated with either of these two chemicals during cell activation in order to promote the accumulation of detectable protein levels within the cell. These are often essential reagents that allow intracellular detection of proteins that would otherwise be in too low in abundance to detect. Monensin is known to block the transport from the medial to the trans cisternae of the Golgi stack. Brefeldin A has been reported to block protein transport from the ER. Specific information about the use of Monensin and Brefeldin A can be found on their respective technical Data Sheets.
Q: Can I vortex my antibody or recombinant protein?
We do not recommend vortexing protein containing solutions (such as recombinant proteins or antibodies) as this could alter the functionality of the product. During shipping, it is possible that liquid is dispersed throughout the tube. Prior to use, quick spin the antibody vial to recover the maximum volume.
Q: Can I vortex samples that have been stained with conjugated antibodies?
Yes, you can but keep it short (pulse vortex), as it is always best to maximize the health of your cells.
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Q: What is the difference between eFluor® Organic Dyes and eFluor® Nanocrystals (NC)?
The eFluor Organic Dyes (eFluor 450, APC-eFluor 780, PerCP-eFluor 710, eFluor 710) are conventional fluorochromes. In contrast, the eFluor nanocrystal line of products is nanocrystals/Quantum dots.
Q: Can eFluor Organic Dyes and eFluor Nanocrystals be used in combination with other organic fluorochromes?
Yes, both the eFluor Organic Dyes and eFluor Nanocrystals are fully compatible with conventional fluorochromes and can be used in the same manner as the dyes that they are replacing. We recommend the eFluor NC Flow Cytometry Staining Buffer cat# 00-3222 when using eFluor nanocrystals either on their own or in combination with organic fluorochromes.
Q: What buffers are compatible with the eFluor® Organic Dyes and eFluor Nanocrystals?
The eFluor Organic Dyes can be used with flow staining buffers that are compatible with conventional fluorochromes.
We have determined that staining of cells in the recommended eFluor NC Flow CytometryStaining Buffer (cat. 00-3222) provides the most consistent results with the eFluor Nanocrystals. While we do see staining in PBS based-buffers, we observe slightly brighter signal intensity when using eFluor NC Flow Cytometry Staining Buffer with the eFluor Nanocrystals.
Q: Can the eFluor® Organic Dyes and eFluor Nanocrystals be used for intracellular staining?
Yes, the eFluor Organic Dyes and eFluor Nanocrystals can be used for intracellular staining. As with the dyes they are replacing, we do recommend a minimum fixation time and data collection as soon as possible for optimal signal when using the eFluor Organic dyes. For information regarding the use of eFluor Nanocrystals for intracellular staining, please refer to the Intracellular staining protocol in our Best Protocols section.
Q: I am unable to analyze my cells stained with eFluor® Organic Dyes or eFluor Nanocrystals today. What options do I have?
Your options will depend on the samples you are analyzing.
If cell viability is not critical, you can store your stained samples at 4°C or on ice overnight in the dark and analyze the following day.
Fix the samples for 30 minutes in 2% formaldehyde in PBS. For fixation we recommend that cells be suspended in 100 µl of eFluor NC Flow Cytometry Staining Buffer and 100 µl of eBioscience IC Fixation Buffer cat. 00-8222, incubated for 20-30 minutes and then washed 1-2X with eFluor NC Flow Cytometry Staining Buffer (cat. 00-3222). The fixed cells should then be stored at 4°C in the dark until analyzed.
Q: Why are you replacing APC-Cy7 and APC-Alexa Fluor® 750 with APC-eFluor® 780, Pacific Blue® with eFluor® 450 and Alexa Fluor® 647 with eFluor® 660?
Due to the dimness, compensation and photostability issues associated with APC-Cy7, we are replacing this fluorochrome with the APC-eFluor® 780 tandem dye. This newly released tandem dye will also be replacing the APC-Alexa Fluor® 750 fluorochrome. The APC-eFluor® 780 tandem dye stains with a similar or brighter staining intensity as APC-Cy7 and APC-Alexa Fluor® 750.
Similarly, the eFluor® 450 will replace our Pacific Blue® fluorochrome offering and eFluor® 660 will replace our Alexa Fluor® 647 offering.
Q:Can the eFluor® Organic Dyes be frozen?
As with other organic fluorochromes, we do not recommend the eFluor® Organic Dyes be frozen.
Q: Are the eFluor® Organic Dyes photo-labile?
As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.
Q: What is the excitation/emission wavelength for the eFluor® Organic Dyes?
As with all eFluor products, the eFluor Organic Dyes are named for their emission wavelength. For information regarding the peak excitation and emission wavelengths for the eFluor® organic dyes, please see our Fluorescent Dyes for Flow Cytometric Analysis.
Q: Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?
Yes, in house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.
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Q: How do eFluor® 60NC, eFluor® 625NC and eFluor® 650NC conjugated antibodies compare to other fluorochrome conjugated antibodies?
A: eFluor® Nanocrystals offer several distinct advantages:
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Narrow emission spectra results in less spectral overlap; thus, less compensation is required
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Excellent signal to noise ratio
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Superior photostability compared to conventional fluorochromes
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Compatible with other fluorochromes thereby allowing more flexibility in multiparameter flow cytometry
Q: Are there special considerations I need to take into account when using the eFluor 605NC, eFluor 625NC, and eFluor 650NC conjugated antibodies?
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Staining buffer - For the optimal staining intensity we recommend the use of the eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222) instead of a PBS-based buffer or eBioscience Flow Cytometry Staining Buffer (cat. 00-4222).
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Intracellular staining - Please see the Intracellular Staining protocol as found in the BestProtocols® section for information regarding intracellular staining.
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Long term fixation - We do not recommend long-term fixation, but rather we recommend a fixation time of 30 minutes in 2% formaldehyde. For fixation we recommend that cells be suspended in 100 µg of eFluor NC Flow Cytometry Staining Buffer and 100 μl of eBioscience IC Fixation Buffer cat. 00-8222, incubated for 20-30 minutes and then washed 1-2X with eFluor NC Flow Cytometry Staining Buffer (cat. 00-3222). The fixed cells should then be stored at 4°C in the dark until analyzed.
Q: Which combination of fluorochromes can I co-stain with eFluor 605NC, 625NC, and 650NC conjugated antibodies?
eFluor nanocrystal reagents can be used in combination with all other fluorochromes in our catalog. As with all fluorochromes, the instrument configuration will determine the fluorochrome use in the design of a multicolor staining panel. Please note that eFluor 605NC and eFluor 625NC reagents cannot be used in the same sample nor can they be used in a staining combination with a PE-Texas Red conjugate as the emission spectra are too similar to resolve. Also be aware that eFluor 650NC reagents will require compensation out of the APC detector.
Q: Can I use eFluor 605NC, 625NC, or 650NC conjugated antibodies instead of Pacific Orange®?
The eFluor 605NC, 625NC, and 650NC conjugated antibodies can be used as an additional fluorochrome off the violet laser. Please refer to question below which indicates appropriate filters to use.
Q: Can I use the eFluor 605NC, 625NC, or 650NC conjugated antibodies together in the same sample?
The eFluor 605NC and eFluor 625NC directly conjugated antibodies cannot be used in combination with one another. The emission spectra are close enough to each other that the available filers cannot distinguish between the two. However, the eFluor 605NC and eFluor 650NC work well together.
Q: Which channels will require compensation with eFluor 605NC, 625NC, and 650NC conjugated antibodies?
In general, the emission spectra of eFluor Nanocrystal conjugated antibodies are narrow, therefore resulting in minimal spill over into other detectors and thus requiring minimal compensation. The filters used in other detectors will determine the amount of compensation that may be necessary. Please be aware that eFluor 650NC will require compensation out of the APC channel.
Q: How does the protocol for staining with eFluor 605NC, 625NC and 650NC conjugated antibodies differ from staining with conventional organic fluorochrome conjugated antibodies?
The eFluor Nanocrystals conjugated antibodies may be used in the same fashion as other fluorochromes, although we recommend the use of the eFluor NC Flow Cytometry Staining Buffer (cat. 00-3222) for optimal performance. eFluor NC Flow Cytometry Staining Buffer is fully compatible with other fluorochromes. The eFluor NC directly conjugated antibodies are provided as “test” sizes pre-diluted to the optimal concentrations for ease of use. Please refer to the product Technical Data Sheet.
Q: How do I prepare the eFluor® NC Flow Cytometry Staining Buffer?
eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222) is supplied as a 5X concentrated stock solution. The 5X stock solution should be diluted with distilled water to a 1X solution. The eFluor® NC Flow Cytometry Staining Buffer does not contain a source of protein and therefore, should be supplemented with 3% Fetal Bovine Serum (final concentration) or equivalent protein prior to use. The buffer should be stored at 4oC.
Q: Which laser do I use to detect eFluor 605NC, 625NC, and 650NC conjugated antibodies?
eFluor Nanocrystal conjugated antibodies are excited by the 355nm (UV), 405nm (Violet) and to a lesser extent the 488nm (Argon) and 561nm lasers. We recommend using the 355nm or 405nm laser.
Q: Which filter sets do I need to detect eFluor 605NC, 625NC or 650NC conjugated antibodies and where do I place the filters?
Table 1: Recommended Filters for Flow Cytometry
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Recommended Filters for Flow Cytometry
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| Fluorochrome |
Dichroic Pass Filter |
Bandpass Filter |
Alternative Bandpass Filters |
| eFluor TM 605NC
|
595 LP
|
605/40
|
605/50, 605/20, 610/20
|
| eFluor TM 625NC
|
595 LP
|
605/50
|
605/40, 625/20
|
| eFluor TM 650NC
|
630 LP
|
660/40
|
|
Placement of filter sets will depend on your current instrument/filter configuration. Please contact eBioscience Technical Support for further details.
Q: Where can I purchase specific filter sets suitable for use with eFluor 605NC, 625NC and 650NC conjugated antibodies?
Filter sets can be purchased from Chroma Technology, Omega Optical, or Semrock, Inc.
Q: Can I fix my cells after staining with eFluor 605NC, 625NC, and 650NC conjugated antibodies? How long can fixed cells be stored prior to analysis?
Formaldehyde fixatives have been found to be compatible with eFluor Nanocrystal conjugated antibody staining. We have determined that short incubations (<30 min) of stained cells in 2% formaldehyde causes only a minor decrease of the fluorescence signal. When optimal staining sensitivity is required, we do not recommend fixing samples. For fixation we recommend that cells be suspended in 100 µl of eFluor NC Flow Cytometry Staining Buffer and 100 µl of eBioscience IC Fixation Buffer (cat. 00-8222), incubated for 20-30 minutes and then washed 1-2X with eFluor NC Flow Cytometry Staining Buffer (cat. 00-3222). The fixed cells should then be stored at 4°C in the dark until analyzed.
We have found the fluorescent signal to be quite stable. Cells which were stained and then fixed have been stored at 4°C up to several days without additional loss (from that observed in the fixation process) in fluorescent signal.
Q: Is staining whole blood samples compatible with eFluor 605NC, 625NC, and 650NC conjugated antibodies?
Samples of whole blood collected directly into Na-Citrate buffer or EDTA can be stained prior to red blood cell (RBC) lysis. It is recommended to wash and resuspend cells in eFluor NC Flow Cytometry Staining Buffer (cat. 00-3222).
Q: When designing a multi-color panel for flow, what I can do to minimize issues with compensation?
When there is significant emission overlap between two fluorochromes, it is recommended that the fluorochromes are conjugated to antibodies against markers that stain independent cell populations to obtain optimal resolution. This is true for fluorochrome combinations such as APC/eFluor 650NC, PE-Cy5 /APC, and Alexa Fluor 700/APC-eFluor 780 (or APC-Alexa Fluor 750). Please contact Technical Support tech@ebioscience.com for further details.
Q: Are eFluor Nanocrystals toxic?
All eFluor Nanocrystals, except eFluor 700NC which has the non-toxic InGaP composition, contain trace amounts of the heavy-metal Cadmium (Cd), in addition to Zinc (Zn) and Selenium (Se). Cadmium is potentially harmful and should be handled with gloves. We recommend that eFluor Nanocrystals be handled in accordance with laboratory practices for heavy metals and users wear appropriate personal protective equipment at all times. These low levels of Cadmium are not likely to pose health risks, however prolonged exposure studies have not been conducted. For further information, please view the Material Safety Data Sheet (MSDS) for these products available on our website.
Q: How do I dispose of eFluor Nanocrystals?
The eFluor Nanocrystals, except those with the non-toxic InGaP composition, are particles composed of a Cadmium core. We recommend their disposal in accordance with your local, state, and federal regulations for reagents containing heavy metals. Please contact your institution’s health and safety officers in order to ascertain the proper disposal of this material. For further information on the composition of these materials, please view the Material Safety Data Sheet (MSDS) available on our website.
Q: Are the eFluor® Nanocrystal conjugated molecules sensitive to light?
As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.
Q: What buffers are compatible with the eFluor® Nanocrystal conjugated molecules?
We have determined that staining of cells in the recommended eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222) provides the most consistent results with the eFluor® Nanocrystals. While we do see staining in PBS based-buffers, we observe slightly brighter signal intensity when using eFluor® NC Flow Cytometry Staining Buffer with the eFluor® Nanocrystals.
Q: When performing flow cytometry, can I fix the cells after staining with the eFluor® Nanocrystal conjugated antibody?
We do not recommend long-term fixation, but rather a fixation time of 30 minutes in 2% formaldehyde. For fixation we recommend that cells be suspended in 100 µL of eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222)and 100 µL of eBioscience IC Fixation Buffer (cat. 00-8222), incubated for 20-30 minutes and then washed 1-2 times with eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222). The fixed cells should then be stored at 4°C in the dark until analyzed.
Q: When performing flow cytometry, is there a recommended starting concentration?
We recommend trying 2 fold dilutions starting at 1:125 and stopping at 1:2000 in 100 µL staining volume. Cell concentrations can vary from 106to 108 cells.
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Q: Is there a particular flow cytometer required for the use of FlowCytomix™ Kits?
FlowCytomix™ microspheres are detectable in most commonly used flow cytometers. The flow cytometer needs to be equipped with one laser (488 nm or 532 nm) capable of detecting and distinguishing fluorescence emissions at 575 nm and far red (685 nm - 690 nm).
FlowCytomix™ Kits have been tested for use on:
EPICS® XLTM / XL-MCL™*
Cytomics TM FC500*
BD FACScan™
BD FACSCalibur™
BD FACSCanto™
FACSCanto™ II
BD TM LSR I
BD TM LSR II
Guava EasyCyte Plus
MACSQuant®
* For the most commonly used flow cytometers (e.g. Becton Dickinson or DakoCytomation) you can immediately start cytometer setup as below.
If you run a FlowCytomix™ assay on a FC500 Instrument from Beckman Coulter you must ensure that the Forward Scatter (FS) measurements are collected at 1-8°. To accomplish this you must insert the FS 1-8° Field Stop into place: remove the front cover to locate the FS 1-8° Field Stop. Slide the knob from the right (= default 1-19° position) to the left and push to lock in place.
On the XL Series systems the EPICS XL/XL-MCL FS Low Angle Collection Kit is recommended to accomplish this.
Q: Is special software required?
Yes, the flow cytometer raw data files should be analyzed using the FlowCytomix™ Pro software. The FlowCytomix™ Pro 2.3 Software is free and can be downloaded at www.bendermedsystems.com/software-download
Q: Does the FlowCytomix™ Pro software also run on Apple Macintosh?
Yes, you can run FlowCytomix™ Pro software on Apple Macintosh with Mac OS X or later.
Q: Does the Bender MedSystems FlowCytomix™ Pro software interact with the flow cytometer operating system?
FlowCytomix™ Pro Software has been successfully operated in conjunction with all commonly used flow cytometer models. It does not interfere with the flow cytometer operating system.
In addition, FlowCytomix™ Pro Software does not need to be installed on the flow cytometer working station; data files can be transferred to any other computer.
Q: What if I want to design my own multiplexing experiment?
FlowCytomix™ Simplex Kits are available for this purpose. Although FlowCytomix™ Simplex Kits are designed for the detection of one specific cytokine, they can be combined with each other provided the beads are compatible as described here:Possible Combinations
Q: When is a FlowCytomix™ Basic Kit required?
Basic Kit is required for the successful use of Simplex Kits. In order to run a FlowCytomix™ assay some reagents are needed only once, even in the case of combining several Simplex Kits. These reagents are provided in a Basic Kit.
For mouse and rat Simplex Kits the mouse/rat Basic Kit is required, cat. no. BMS8440FF.
For human Simplex Kits PE (E-selectin, ICAM-1, ICAM-3, PECAM-1, P-selectin, VCAM-1) the human Basic Kit PE cat. no. BMS8421FF is required. In case Simplex Kits and Simplex Kits PE are combined, the human Basic Kit is required.
For all other human Simplex Kits the human Basic Kit cat. no. BMS8420FF is required.
Q: Are FlowCytomix™ assays performed in plates or in tubes?
The FlowCytomix™ assay can be performed like a conventional ELISA in a 96-well filter plate or in individual tubes . When using a 96-well filter plate, a filtration manifold is required (cat. no. BMS497FF). The use of tubes does require centrifugation of the tubes during the washing steps.
Q: Why is a cytometer setup required before measuring FlowCytomix™ standards and samples?
The cytometer setup is required to prepare the instrument for the bead-based assays. FlowCytomix™ setup beads are included in the Basic Kits and in the Multiplex Kits to prepare the correct instrument settings.
With every new experiment, before starting the acquisition of standards and samples, stay in SET UP MODE and adjust the settings using the highest standard (Standard 1).
Q:What wavelength is used for measurement?
The maximum emission of PE is at 578 nm and the emission of the beads is in the far red region (685 - 690 nm).
Q: Is it possible to measure with high "flow through" settings?
There are often three flow through settings: low, medium, & high.
Increasing the "flow through" rate may result in a dispersed bead population. Therefore, it is recommended to start with the low "flow through". The rate can be increased if the bead population does not start to disperse.
Once the final instrument settings are saved, do not change the "flow through" rate during measurement.
Q: Does a FlowCytomix™ Kit work on a Luminex™ instrument?
No, FlowCytomix™ Kits can only be measured on a flow cytometer.
Q: Is it correct to use the non-linear part of the standard curve during analysis?
Yes, it is possible to use the non-linear part of the standard curve for calculation of results. Dilutions of samples behave in the same way as the standard curve.
Q: How many analytes can be combined in one assay?
With FlowCytomix™, up to 20 analytes are possible.
Q: Which analytes can be combined?
Up to 20 FlowCytomix™ Simplex Kits within one species can be combined.
FlowCytomix™ Simplex Kits with an identical bead population cannot be combined. For possible combinations, please click here: Possible Combinations.
Q: Can FlowCytomix™ Multiplex Kits be combined with each other?
All FlowCytomix™ Human Multiplex Kits can be combined with BMS810FF human Th1/Th2 11plex. All FlowCytomix™ Mouse Multiplex Kits can be combined with BMS820FF Mouse Th1/Th2 10plex. A combination of BMS811FF Human Cardiovascular Panel with BMS812FF Human Adhesion Panel is possible for some analytes which do not use the same bead sets.
Q: Can FlowCytomix™ Simplex Kits be combined with a Multiplex Kit?
Yes, FlowCytomix™ Human Simplex Kits can be combined with Human Multiplex Kits provided the beads are compatible as described here: Possible Combinations
Q: Can FlowCytomix™ components from different lots be mixed?
The FlowCytomix™ bead sets, standards and conjugates are lot-specific and must be used in combination with each other. Do not mix these components from different kit lots.
FlowCytomix™ Assay Buffer, Reagent Dilution Buffer and Streptavidin-PE are not lot-specific and can therefore be exchanged between different kit lots.
Q: Is it possible to combine FlowCytomix™ Biotin Conjugates with PE-conjugates in one conjugate mixture?
Yes. However, if you are using at least one Biotin-Conjugate in your combination a further incubation step with Streptavidin-PE is necessary. This does not interfere with the PE-conjugates.
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Q: What do Platinum ELISA Kits contain?
All reagents necessary to perform 1 or 10 plate ELISA assays, including optimized capture-antibody pre-coated plates.
Q: Which samples are suitable for Platinum ELISA Kits?
Most ELISAs are suitable for serum, plasma, cell culture supernatants and other body fluids. Detailed information is given in the product manual for each individual kit.
Q: Do I have to run the Platinum ELISA standards and samples in duplicates?
No, but it is highly recommended to make at least double determinations of each sample and standard point.
Q: Is it possible to use the reagents of other Platinum ELISA kits/lots?
The reagents within the Platinum ELISA kits are not interchangeable between different lots of the same kit or between kits against different analytes.
Q: Can I always use the same manual for different ELISA kit lot numbers?
We recommend that you use the manual included with the Platinum ELISA kit.
Q: Is it necessary to wash the Platinum ELISA plates before usage?
An initial washing step is not necessary but recommended as accuracy may be increased.
Q: Are shorter or longer incubation times possible?
Incubation times should be followed exactly as stated in the product manual to ensure optimal Platinum ELISA test performance.
Q: Can I omit the dilution of my Platinum ELISA samples with assay buffer/sample diluent when performing the assay?
We recommend following the instructions for sample dilutions as provided in the manual. We cannot guarantee optimal performance of the assay if internal assay dilutions as instructed were omitted.
Q: What if it does not look like the supplied reagent volume is sufficient for running the Platinum ELISA?
Q: What if it does not look like the supplied reagent volume is sufficient for running the Platinum ELISA?
In case of small reagent volumes, please spin down vial before use to make sure to all contents are collected in the bottom of the tube.
Q: Is it possible to store the reagents other than indicated?
Performance of the Platinum ELISA cannot be guaranteed if reagents are not stored as indicated.
Q: Do you have information regarding the normal values detectable with your Platinum ELISA Kit?
You can find expected "normal levels" listed in the Platinum ELISA Kit manual under "Expected values".
Please be aware that these normal values were obtained in our laboratories with a limited number of samples. We strongly suggest that each laboratory perform studies in order to establish normal range for their sample populations.
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Q: How many tests can I perform with an Instant ELISA® Kit?
With an Instant ELISA®, 128 tests (includes samples plus standards) can be performed. The number of tests results from the following composition:
• One 96 well plate designed only for samples; 96 samples
• Two 8-point standard curve in duplicates; 32 wells; Thus the kit can be used at two different time points, each with their own standard curve.
Q: Do I have to add recombinant standard to the wells?
No, the recombinant standard is included as a component of the lyophilized reagents in the wells.
Q: Do I have to add the secondary detection antibody to the wells?
No, the secondary antibody is included as a component of the lyophilized reagents in the wells.
Q: Do I have to dilute my samples prior to adding them to the wells?
In general, one can use undiluted samples with the Instant ELISA. Please refer to the product manual to determine if dilution of the sample is required for your kit of interest. The volume of sample required for the individual kits will also be listed in the product manual.
Q: Is the reconstitution volume of the standards the same for different lot numbers?
For some Instant ELISA® kits, the reconstitution volume of the standard and blank wells may differ (these components are lot specific). Please use the manual provided in the Kit for specific volumes to use.
Q: How do I calculate my samples?
The calculation factor is always stated in the manual.
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Q: Can the amplification reagents be prepared ahead of time?
No, the amplification reagents have to be prepared immediately before use. The reagent AR1 contains methanol which evaporates easily.
Q: Can I deviate from the recommended incubation times?
No, after adding the amplification reagents to the sample wells, the incubation times have to be followed exactly for optimal use.
Q: Can I perform the incubations at higher temperatures?
Incubations should occur at ambient temperatures not higher than 25°C.
Q: Are the wash steps necessary?
Yes, please follow the washing steps exactly as outlined in the product manual (for optimal readings: an automatic plate washer is recommended).
Q: How should I prepare the standards for use with the High Sensitivity Kits?
We recommend that you prepare the standard in eppendorf tubes.
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Q: What do the Ready-Set-Go! ELISA Sets contain?
• Capture Antibody: Pre-titrated, purified antibody
• Detection Antibody: Pre-titrated, biotin-conjugated antibody
• Standard: Recombinant cytokine for generating standard curve and calibrating samples
• ELISA Coating Buffer Powder
• Assay Diluent: 5X concentrated
• Detection enzyme: pre-titrated Avidin-HRP
• Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
• Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
• 96 Well Plate: Corning Costar 9018 (included with product Cat. #’s ending in suffixes -22, -76, -86)
Q: What are the storage conditions for your Ready-Set-Go! ELISA Sets?
All of the reagents are to be stored at 4°C upon receipt, except for the recombinant standards. The recombinant standards MUST be stored at or below -80°C to ensure stability and performance.
Q: What is the expiration date for your Ready-Set-Go! ELISA Sets?
The majority of our Ready-Set-Go! ELISA Sets are guaranteed for one year from the date of receipt. However, we do have several sets that are guaranteed for 6 months upon date of receipt. Please refer to the Certificate of Analysis for shelf-life information.
Q: What is the sensitivity of your Ready-Set-Go! ELISA Sets?
The sensitivity ranges of our Ready-SET-Go! ELISA sets vary depending on the cytokine/protein being measured. Please refer to the product datasheet for set specific information.
Q: What concentrations should I use for my recombinant standard?
Each Ready-Set-Go! ELISA Set comes with its own Certificate of Analysis (CofA). The directions on the CofA should be strictly followed to ensure performance of the reagents. The CofA gives specific instructions on how to dilute each component of your lot specific set. The instructions on the CofA should not be deviated from in order to ensure accurate results.
Q: I want to collect my tissue culture supernatant daily for a time-course study and run all of my samples at once by ELISA. How should I store my supernatants?
The supernatants can be harvested and stored -20°C. Avoid freeze/thaw cycles. It is recommended to briefly centrifuge samples after collection to pellet any dead/floating cells remaining in the supernatant. Transfer to new tube for storage.
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Q: Do you offer antibody pairs for use in an ELISA?
Please see our Antibody Pairs Chart for a complete listing of the capture and detection antibodies, and recombinant protein standards available.
Q: What other reagents will I need to run an ELISA using the antibody pairs?
eBioscience provides the following components needed to run a successful ELISA. They include the following:
• High-affinity binding NUNC Maxisorp p (cat. 44-2402) or Corning Costar 9018 ELISA Plates (cat. 44-2504)
• Coating Buffer
• 5X Assay Diluent ( (cat. 00-4202)
• Avidin-HRP P (cat. 18-4100)
• Super AquaBlue ELISA substrarate (cat. 00-4203) or TMB (cat. 00-4201)
For ELISA pairs, eBioscience recommends using the HRP compatible substrate, Super AquaBlue (read at 405nm). Our Ready-Set-Go! ELISA Sets include TMB substrate (read at 450nm).
Q: What are the differences between TMB and Super AquaBlue?
TMB is a faster developing, stronger substrate that yields greater amplification/sensitivity/background. The use of an acid stop solution produces a yellow end product read at 450nm. Super AquaBlue is a slower developing blue/green substrate particularly useful for kinetic studies.
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Q: What are the components of the Ready-Set-Go! ELISPOT Sets?
• Capture Antibody: Pre-titrated, Functional Grade (low endotoxin) purified antibody
• Detection Antibody: Pre-titrated, biotin-conjugated antibody
• ELISPOT Coating Buffer
• Assay Diluent: 5X concentrated
• Detection enzyme: pre-titrated Avidin-HRP
• Certificate of Analysis: Lot-specific instructions for dilution of antibodies and enzyme
Q: Do your antibody pairs for ELISA work in ELISPOT as well?
Most, but not all, of our ELISA antibody pairs work for ELISPOT. We continue to evaluate and optimize our anti-human and anti-mouse cytokine antibody pairs for ELISPOT applications. Please see our ELISPOT BestProtocols® page for a detailed protocol and a reference table with the list of antibody pairs that can be used in ELISPOT.
Q: I recently bought one of your Ready-Set-Go! ELISA sets, can I use the antibody pairs to run an ELISPOT?
We do not recommend the use of our ELISA kits for the ELISPOT application. We cannot guarantee the successful use of our ELISA kits in an ELISPOT application.
Q: What are the critical parameters for successful ELISPOT assay?
It is critically important to use a high affinity binding PVDF membrane plate, rather than regular ELISA plate or nitrocellulose membrane plate. Additionally, highest affinity antibodies for capture work best in this assay. Empirical optimization of conditions such as cell density, stimulus/mitogen, and kinetics is necessary.
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Q: What types of recombinant proteins does eBioscience offer?
The majority of the recombinant proteins eBioscience offers are cytokines and chemokines. However, we do offer an expanding list of various factors (growth, stimulating, etc.) as well.
Q: What formats do you offer?
eBioscience offers recombinant proteins, as well as Carrier Free recombinant proteins. The recombinant proteins are provided in a sterile buffer (formulation on individual Technical Data Sheets) with the addition of a carrier protein, which in most cases is 1% BSA. The Carrier Free recombinant proteins are provided in a sterile buffer (formulation on individual Technical Data Sheets) with no additional proteins or preservatives.
Q: What is the shelf life of your recombinant proteins?
The majority of eBioscience recombinant proteins have a guaranteed shelf life of one year unless indicated on our Technical Data Sheets. This guarantee is provided if that they are kept under optimal storage conditions as stated on the Data Sheet (at -80°C). If you are not completely satisfied with the performance of your product, please contact technical support at tech@ebioscience.com for assistance.
Q: What are the concentrations of the recombinant proteins offered?
In most cases, the recombinant proteins are offered at a concentration of 0.1mg/mL, while the carrier free recombinant proteins are offered at 0.5mg/mL. However, we do recommend you refer to the product vial for lot specific information.
Q: How should I store my recombinant protein(s)? Can I dilute them upon receipt?
You will want to store the recombinant proteins at or below -80°C. The proteins can be diluted, and specific information regarding this is available on the Technical Data Sheets. However, key considerations are as follows:
1. always quick spin the vials prior to opening
2. dilute primary stock to a minimum of 10ug/mL
3. perform dilutions in a buffered saline (formulation on technical data sheet) which contains 1.0% BSA or 10% FBS as a protein carrier/stabilizer
4. make aliquots to minimize the number of freeze/thaw cycles. Avoid freezers with thermal cycling, such as frost free freezers.
Q. What is the specific activity of eBioscience recombinant proteins?
The specific activity (Units/mg) for each recombinant protein is available on the Technical Data Sheet under Bioactivity. The information can also be found in our Bioassays- Recombinant Cytokines and Recombinant Growth Factors Quick Guide on our BestProtocols® page.
Q. What is meant by a ‘unit’ of protein activity?
A "unit” is defined as the concentration of protein required to induce half maximal activity (e.g. in ng/mL). This is also referred to as the ED50, or 50% effective dose. This method of expressing potency should only be used for proteins whose dose response curves are sigmoidal in shape (e.g. not chemotaxis assays).
Q:What is the relationship between the specific activity (Units/mg) and ED50 of a protein?
The formula for converting the activity as an ED50 in ng/mL to a specific activity in Units/mg is:
Specific activity (Units/mg) = 106/ ED50 (ng/mL)
Q: Does each lot of recombinant protein have the same specific activity?
No, each lot can vary slightly with regards to its specific activity. The information on the Technical
Data Sheet for each product is provided as a guide. For lot specific information, please contact eBioscience Tech Support.
Q: What is the difference between laboratory (observed) units and international units?
Laboratory units are the actual values obtained from running an assay with a particular protein in an assay in your lab; i.e., the activity (ED50) you observe on your target cells. ‘International’ units are consensus values of potency derived from a collaborative NIBSC effort to standardize reported use of proteins. These values are derived from bioassay testing of the same protein by many target cell types/substrains. It is very likely that the ‘laboratory’ units you observe and the NIBSC values will not correlate 1:1; e.g., it might take 0.1 - 20 U/ml to see 1 U/ml in your experiment. These are bioassay standards describing potency in bioassay only. The mass values assigned to these are not hard values and use of these for immunoassay standardization is of limited value unless assays calibrated by the NIBSC standard use the same capture and detection antibody clones.
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Q: Can I use eBioscience antibodies for immunoblotting (WB)?
We do not routinely test our antibodies in house by Western blotting (WB) unless it was specifically developed for that purpose; therefore, we rely on the literature as the basis for reporting WB as an approved application for a given clone. If a product is suitable for WB, it will be noted under reported applications on the products technical data sheet. Please refer to the Product Technical Datasheet for more information.
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Q: Can I use eBioscience antibodies for immunoprecipitation (IP)?
We do not routinely test our antibodies in house by immunoprecipitation (IP) unless it was specifically developed for that purpose; therefore, we rely on the literature as the basis for reporting IP as an approved application for a given clone. If a product is suitable for IP, it will be noted under reported applications on the products technical data sheet.
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Q: I am interested in staining tissue sections. Are your antibodies suitable for this application?
We are in the process of testing our antibodies in house on IHC applications. Please contact eBioscience Tech Support for additional information that may be available.
Q: Do you have protocols available for your antibodies and immunohistochemical staining?
We do have general IHC protocols available for frozen tissues and paraffin embedded tissues. For clone specific information, please contact eBioscience Tech Support.
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Q: When performing immunofluorescent staining on cells and tissues, are there any changes to the protocol?
Please refer to our recommended protocols in the Best Protocols section of our website.
Q: My staining works fine with purified followed by a secondary antibody, but the eFluor® Nanocrystal conjugated antibody does not appear to be working. What could be wrong?
Although the protocol works for two-step or three-step staining, the conditions may need to be changed for staining with eFluor® Nanocrystal conjugates. Here are several points to consider:
1. Protocol- We recommend using the protocols developed by eBioscience that have been shown to work for a variety of direct eFluor® Nanocrystal conjugated antibodies. Please refer to our recommended protocols in the BestProtocols section of our website.
2. Blocking buffer- We have formulated two versions: one containing high amounts of protein and another with lower levels. We recommend starting with IHC/ICC Blocking buffer-Low Protein (Cat. No. 00-4953), which has been shown to be optimal for general use when staining cytoskeletal, cytoplasmic and membrane proteins. For improved specificity and better signal-to-noise ratio, the IHC/ICC Blocking Buffer- High Protein (Cat. No. 00-4952) has been found to give superior results especially with antibodies to nuclear antigens and FFPE tissue.
3. Incubations times- For best results, we recommend blocking non-specific sites for 1 hour at room temperature and incubating eFluor® Nanocrystal conjugates overnight in blocking buffer at 2-8°C protected from light.
4. Mounting media- Please refer to table below on selection of mounting media by application.
Q: Will eFluor® Nanocrystal conjugated antibodies blink?
Although nanocrystals can blink, all efforts are taken during manufacturing to prevent this.
Q: What should my starting concentrations be?
All antibodies should be titrated for optimal performance. The final antibody conjugate concentration from both the Amine and Sulfhydryl-Reactive kits should be in the range of 3-6 µM for the eFluor® Nanocrystal 605 and 650 conjugates. For ICC and IHC applications, we recommend a starting concentration of 1-40 nM, although the optimal concentration must be determined for the cells or tissue of interest.
Q: How should I mount my slides?
Special Note: For experiments requiring extended illumination times or using organic dyes in combination with eFluor® Nanocrystal conjugates, it may be necessary to mount stained tissue or cells with mounting medium containing an anti-fade component. We suggest using Vectashield mounting medium for these conditions.
Q: How should I store my slides?
Slides should be sealed with clear nail polish and stored at 2-8°C in the dark.
Q: How long do I have to look at my slides after completing the staining?
A benefit to using the nanocrystals is that they do not photobleach like organic dyes. If mounted and stored properly, the slides will be good for weeks, if not longer.
Q: Which buffers and detergents are compatible with the eFluor® Nanocrystal conjugated antibodies?
Because you are working with directly conjugated antibodies, there should not be a need for detergents in your buffers. It is best to avoid their use if possible. We recommend using Tris Buffered Saline (TBS), pH 7.4 (Cat. No. 00-4954) rather than Phosphate buffered saline (PBS) for washing following binding of eFluor® Nanocrystal conjugated antibodies.
Q: Can I use a PAP pen?
No, we find that traditional PAP pens are not compatible with eFluor® Nanocrystal conjugated antibodies.
Q: What filters sets do I need to detect the eFluor nanocrystals for microscopy?
|
Optimal filter sets for Fluorophores
|
| eFluor® Nanocrystals |
Target Emission |
Excitation filter/Bandwidth |
Dichroic Beamsplitter |
Emission filter/Bandwidth |
| eFluor 525NC
|
525
|
460, 425/45, 415/100
|
505
|
520/20
|
| eFluor 565NC
|
565
|
460, 425/45, 415/100
|
470
|
565/20
|
| eFluor 605NC
|
605
|
460, 425/45, 415/100
|
470
|
600/20
|
| eFluor 625NC
|
625
|
460, 425/45, 415/100
|
470
|
620/20
|
| eFluor 650NC
|
650
|
460, 425/45, 415/100
|
470
|
655/20
|
| Fluorophores |
Target Emission |
Excitation filter/Bandwidth |
Dichroic Beamsplitter |
Emission filter/Bandwidth |
| DAPI/Hoeschst 33342
|
450
|
365/50
|
1
|
450/65
|
| Alexa 488/FITC/GFP
|
535
|
475/40
|
510LP
|
535/45
|
| TRITC/PE/Cy3
|
585
|
546/12
|
470
|
585/40
|
| Texas Red/eFluor 615
|
615
|
560/55
|
585LP
|
645/75
|
| Alexa 647/Cy5/Draq5
|
700
|
620/60
|
665LP
|
700/75
|
For best results use the filter sets listed above. Additional filter sets may be usable for the fluorophore of interest, but are likely to result in decreased signal intensity.
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