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 Preparation

Sonication versus no sonication
Phospho-Histone H3 (Ser10) Antibody #9701

CKR/PAEC cell extracts

Primary Antibody Dilution Buffer

Milk versus BSA
Phospho-Akt (Ser473) Antibody #9271

C2C12 cells + insulin

Primary Antibody Incubation

Overnight at 4°C versus 2 hours at room temperature

Phospho-Akt (Ser473) Antibody #9271

HeLa cell extracts

PKCδ Antibody #2058

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 CST recommended incubations

iBlot^® (quick transfer-Invitrogen) SNAPi.d.™ (quick incubations-Millipore) * determined optimal antibody concentration for SNAPi.d.™