Changes to Recommended Western Blotting Protocols

Cell Signaling Technology (CST) antibodies are validated utilizing our really useful western blotting protocol, developed and optimized by CST scientists. All knowledge proven on product webpages and datasheets was generated utilizing these protocols. We strongly suggest following our protocols to obtain optimum outcomes with all of our antibodies. Below are just a few examples of the results minor adjustments to the usual protocol could have in your outcomes.

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 really useful in CST protocol)
PBS-T (altered protocol)

Transfer and Antibody Incubations

Cell Signaling Technology recommends moist switch (as really useful by producer) adopted by one hour blocking and in a single day main antibody incubation at 4˚C.

iBlot® is a dry blotting system that completes switch in 7 minutes. SNAPi.d.™ is a vacuum operated incubation system, which reduces antibody incubation instances to lower than 30 minutes.

Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb #9215

Wet switch CST
 really useful incubations
iBlot® (fast transfer-Invitrogen) SNAPi.d.™ (fast incubations-Millipore) * decided optimum antibody focus for SNAPi.d.™

iBlot® is a trademark of Invitrogen Corporation.
SNAPi.d.™ is a trademark of Millipore Corporation.