In untreated cell lines or tissues, the basal expression levels of many post-translational modified proteins are low. We recommend using PhosphoSitePlus to search for literature on the use of low throughput methods related to your specific modification site, or using our target positive control processing table to search for processing methods and cell lines or tissues that can serve as positive controls.

Adding protease and phosphatase inhibitors to cell extracts is crucial for avoiding protein degradation and maintaining protein yield. Sodium pyrophosphate (final concentration of 2.5 mM) and β - glycerophosphate (final concentration of 1.0 mM) should be added as serine/threonine phosphatase inhibitors to the lysis buffer. Sodium orthovanadate (final concentration of 2.5 mM) should be added to inhibit tyrosine phosphatase. Protease Inhibitor Cocktail (100X) (# 5871) or Protease/Phosphatase Inhibitor Cocktail (100X) (# 5872) can also be used.

For example, inducing phosphorylation through treatment: using Phospho-IKK α/Β (Ser176/180) (16A6) Rabbit mAb for differentiation with TPA (# 9905, 80 nM, 24 hours), and performing Western blot analysis on the extract of THP-1 cells treated with 1 μ g/mL LPS for the indicated time. Phosphorylation of certain targets such as IKKa/b requires specific processing conditions to achieve optimal expression levels.