Antibody Validation for Flow Cytometry
Our scientists are at the bench daily to produce and validate our antibodies, so they have hands-on experience and knowledge of each antibody’s performance.
Flow cytometry is sensitive, quantifiable, fast and multiparametric. Large numbers of cells can be analyzed quickly for protein expression, DNA content, cell cycle state, cell size, light scatter characteristics, and ionic shifts—even in very small subpopulations. In addition, modern flow cytometers can measure the intensities of five or more fluorescent markers simultaneously, which is important when the supply of cells is limited. By combining flow cytometry with thoroughly validated antibodies from Cell Signaling Technology, it is possible to examine complex intracellular signaling cascades in cell lines, dissociated tissues, aspirates, or hematology specimens. All of our over 550 antibodies validated for flow cytometry, including our fluorochrome-conjugated antibodies, have been screened to determine optimal dilutions and to verify specificity.
Validation Steps Include
- Serial dilution is used to determine optimal dilution.
- Comparison of signal to isotype control is used to estimate the nonspecific binding of primary antibodies.
- Use of known positive and negative cell lines verifies target specificity.
- Treatment of cell lines with pathway-specific inhibitors or activators verifies target specificity.
- The use of blocking peptides, siRNA, and expression vectors verifies specificity of staining.
- Phosphatase treatment confirms phospho-specificity of the antibody.
- Extensive quality control testing guarantees stability over time and eliminates lot-to-lot variability.
- Optimized protocols are provided and dilutions are predetermined.
At CST we routinely validate the performance of our antibodies across multiple platforms. However, if you purchase an antibody from an alternative vendor, it is prudent to test its performance in applications other than flow cytometry, to further ensure specificity. We would recommend doing additional testing using immunofluorescence (IF). Below is an example, which demonstrates why such testing may be necessary.
A comparative analysis of the performance of a phospho-Stat5 antibody from CST (#4322) with a phospho-Stat5 antibody from an alternative vendor. When looking at a cell line positive for phospho- Stat5, both antibodies enable detection. While the non-CST Ab clone gives rise to a brighter signal by flow cytometry (A) (bar graph on the left), which is dampened by imatinib treatment, it exhibits incorrect sub-cellular localization when examined by IF (B). This is particularly evident when cells are treated with EGF to enhance the strength of the signal. This suggests that only the CST antibody clone for phospho-Stat5 detects its target specifically, correctly localizing to the nuclear compartment of the cell.
Flow cytometric analysis of HeLa cells (blue) and HUVEC (green) using CD102/ICAM-2 (D7P2Q) Rabbit mAb #13355. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.