Changes to Recommended Western Blotting Protocols
Cell Signaling Technology (CST) antibodies are validated using our recommended western blotting protocol, developed and optimized by CST scientists. All data shown on product webpages and datasheets was generated using these protocols. We strongly recommend following our protocols to achieve optimal results with all of our antibodies. Below are a few examples of the effects minor changes to the standard protocol may have on your results.
Lysate PreparationSonication versus no sonication 
CKR/PAEC cell extracts |
Primary Antibody Dilution BufferMilk versus BSA 
C2C12 cells + insulin |
Primary Antibody Incubation
Overnight at 4°C versus 2 hours at room temperature
|
Phospho-Akt (Ser473) Antibody #9271 
HeLa cell extracts 
HeLa cell extracts |
Phospho-GSK-3β (Ser9) Antibody #9336 
HeLa cell extracts |
Wash and Dilution Buffer
TBS-T versus PBS-T
Phospho-c-Jun (Ser63) II Antibody #9261
c-Jun (60A8) Rabbit mAb #9165
Ribosomal Protein S3 Antibody #2579
Calnexin (C5C9) Rabbit mAb #2679
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855
TBS-T (as recommended in CST protocol) |
PBS-T (altered protocol) |
Transfer and Antibody Incubations
Cell Signaling Technology recommends wet transfer (as recommended by manufacturer) followed by one hour blocking and overnight primary antibody incubation at 4˚C.
iBlot® is a dry blotting system that completes transfer in 7 minutes. SNAPi.d.™ is a vacuum operated incubation system, which reduces antibody incubation times to less than 30 minutes.
Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb #9215
Wet transfer |
iBlot® (quick transfer-Invitrogen) |
iBlot® is a trademark of Invitrogen Corporation.
SNAPi.d.™ is a trademark of Millipore Corporation.
PhosphoSitePlus®