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Phosphorylation-specific Antibodies Frequently Asked Questions


General Questions

Q. How does eBioscience determine the specificity of their phosphorylation-specific antibodies?

Antibodies are tested and validated in a variety of ways. First, we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation specific staining only in the cells in which the specific pathway of interest has been activated. Second, we verify that phosphorylation specific staining is observed only in cell types in which the protein is expressed and not in cell types in which the protein is not expressed. Third, whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate).

Q. Can total and phosphorylated protein be analyzed in parallel?

In theory, this can be done if the antibodies to both proteins work in the same buffer system and if the antibodies recognize different epitopes. Please refer to the individual Technical Data Sheets for information about buffer compatibility. Also, consider using eBioscience InstantOne ELISA Kits for immunoassay quantitation of both total and phosphorylated proteins such as AKT1/2/3, JNK1/2/3, NFκB, STAT3, and STAT5.

Q. Can I perform phosphorylation specific staining together with transcription factor staining at the same time? If so, what buffer and protocol do I use?

In theory, this can be done if the antibodies against both the transcription factor and the phosphorylated protein work in the same buffer system. For example, Anti-Human/Mouse phsopho-H2AX and all transcription factor antibodies offered by eBioscience will work in the Foxp3/Transcription Factor Buffer System.

Q. Can I perform phosphorylation specific staining and stain for intracellular cytokines at the same time?

In theory, if the buffer systems are compatible for the antibodies of interest, this can be done. In practice, cytokine translation and phosphorylation events do not typically occur in the same timeframe, therefore, it may be difficult to detect both events in the same sample. The results will be depended on the kinetics and should be determined for each system and protein of interest.

Q. What are the appropriate controls to run?

It is important to understand whether the stimulation/treatment results in an upregulation or a down regulation of the phosphorylation event. Thus, it is recommended that changes in phosphorylation levels in stimulated/treated versus unstimulated/untreated cells be compared. A histogram or density plot overlay these two treatments will provide a better assessment of a change in phosphorylation event than using isotype controls.

Q. Can I use other buffer systems from other companies?

In general, you can use other companies’ buffer systems provided that their buffers are of a similar composition as the buffers recommended by eBioscience. Be aware that results will vary depending upon the buffer system used. eBioscience antibodies have been optimized for use in eBioscience buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

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Optimizing for Multiparametric Staining

Q. What is the best way to maintain surface staining when using the methanol-based protocol?

Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450, FITC, and eFluor 660).

Please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. We recommend staining after stimulation/treatment, fixation, and methanol treatment. Please refer to our table (Antibody Clone Performance Following Fixation / Permeabilization) that evaluates the performance of numerous clones in the methanol-based buffer system.

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Protocol General Questions

Sample Preparation:

Q. What cells types can be stained with eBioscience’s protocols?

We have tested many different cell lines (mainly hematopoietic) as well as various mouse tissues (spleen, bone marrow, lymph nodes) and human peripheral blood cells (PBMCs prepared using Ficoll). In some cases, when the phosphorylation-specific antibody uses the Foxp3/Transcription Factor Buffer System (for example, Anti-Human/Mouse phospho-H2AX), refer to the Lysed Whole Blood staining protocol for the Anti-Foxp3 antibody.
Note: An optimized protocol for staining stimulated whole blood is in development; contact Technical Support (tech@ebioscience.com) for updates.

Q. Can I stain adherent cells and if so, what protocol/buffers/cell dissociations buffers do I use?

Adherent cells can be stained. Plate cells as necessary for the experiment desired. Stimulate cells in dishes/plates followed by fixation as necessary for the antibody being used for staining. Note that protocols and buffers are not cell type-specific but rather antibody specific. To dissociate cells, use either gentle scraping or EDTA treatment.

Protocol (buffers and timing):

Q. What are the best protocols/buffers to use for each cell type?

The protocol/buffer is not cell type-dependent (except in the case of whole blood). Rather, each antibody has a protocol and buffer system that is recommended for optimized results. Please refer to the antibody clone specific Technical Data Sheet.

Q. Which buffer should I use with which phosphorylation-specific antibody?

The optimal buffers are noted on the Technical Data Sheet for each antibody.

Q. What is the optimal protocol for phosphorylation-specific antibodies?

Each antibody has been tested in three different buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody.

** Protocols for each buffer system can be found in our Flow Cytometry Intracellular Staining Quick Guides.

Q. How long can samples that have been fixed and placed in methanol be stored? What is the recommended temperature?

At eBioscience, we have data demonstrating that samples stored in methanol at -20°C or at -80°C are stable for several weeks. We do not recommend long-term storage of samples fixed in Foxp3/Transcription Factor Buffer or in IC fixation buffer.

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