We recommend using expression profiling analysis tools such as BioGPS or Human Protein Atlas, as well as scientific literature, to determine whether cells or animal tissues are expected to fully express the target protein of interest. We always recommend setting up known positive controls to assess experimental results. To learn about the recommended control information for many of our antibodies, please refer to the positive control processing table for our targets. 

For whole cell extracts, it is recommended to sample at least 20-30 ug of protein per lane to detect the total amount of target/unmodified target in the whole tissue extract. However, it is usually necessary to increase the total protein loading to at least 100ug per lane to detect modified targets (such as phosphorylation and cleavage targets) in whole tissue extracts. When only a small fraction of cells in the organization contain post-translational modification targets, the whole tissue extract may need to be loaded with more proteins. Adding protease and phosphatase inhibitors to cell extracts is crucial for avoiding protein degradation and maintaining protein yield. We suggest adding leuprorelin (final concentration of 1.0ug/mL) and PMSF (# 8553) as protease inhibitors to the lysis buffer. Protease Inhibitor Cocktail (100X) (# 5871) or Protease/Phosphatase Inhibitor Cocktail (100X) (# 5872) can also be used.