Antibody Validation for Western Blotting
Western blotting remains one of the most common scientific methods for monitoring protein expression in cells or tissue. The accuracy of western blot results relies heavily of the quality of the primary antibody employed in the immunoblotting. Cell Signaling Technology (CST) provides the highest quality primary and secondary antibodies available for western blotting. CST™ antibodies are produced in-house and validated extensively according to a rigorous protocol.
Validation Steps Include
- Examination of several cell lines and/or tissues of known expression levels allows accurate determination of species cross-reactivity and verifies specificity.
- Treatment of cell lines with growth factors, chemical activators or inhibitors, which induce or inhibit target expression, verifies specificity. Phosphatase treatment confirms phospho-specificity.
- The use of siRNA transfection or knockout cell lines verifies target specificity.
- Side-by-side comparison of lots to ensures lot-to-lot consistency.
- Optimal dilutions and buffers are predetermined, positive and negative cell extracts are specified, and detailed protocols are already optimized, saving valuable time and reagents.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® BcL-xL siRNA I #6362 (+) or SignalSilence® Bcl-xL siRNA II #6363 (+), using Bcl-xL (54H6) Rabbit mAb #2764 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Bcl-xL (54H6) Rabbit mAb confirms silencing of Bcl-xL expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
Phosphatase and Activator Treatment
Western blot analysis of extracts from 293, NIH/3T3, and C6 cells, treated with λ phosphatase or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).
Analysis of Multiple Cell Lines
Western blot analysis of extracts from various tissues and cell lines using JIP4/SPAG9 (D72F4) XP® Rabbit mAb #5519.
Western blot analysis of HeLa cells, untreated or treated with IFN-α, comparing lots 1, 2, 3 and 8 of Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145. Note: Signal remains consistent from lot to lot. (Recommended dilution for western blot was changed to 1:2000 with release of lot 3.)
Western blot analysis of EGF Receptor Control Cell Extracts #5634 using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 (upper) and EGF Receptor (D38B1) XP® Rabbit mAb #4267 (lower).