![]() Sodium orthovanadate (2.5 mM final concentration) should be included to inhibit tyrosine phosphatases. Sodium pyrophosphate (2.5mM final concentration) and beta-glycerophosphate (1.0mM final concentration) should be included as serine/threonine phosphatase inhibitors in the lysis buffer. Inclusion of protease and phosphatase inhibitors in the cell extract is essential to avoid protein degradation and maintain protein yield. We recommend using PhosphoSitePlus to look up low-throughput papers referencing your particular modification site or use our Control Treatments by Target table to find an example of a treatment and cell line or tissue that works well as a positive control. Many post-translationally modified proteins are expressed at low basal levels in cell lines or tissues without treatment. Low Level of Phosphorylated or Modified Protein Protease Inhibitor Cocktail (100X) (#5871) or Protease/Phosphatase Inhibitor Cocktail (100X) (#5872) may also be used. We recommend that the lysis buffer include leupeptin (1.0 ug/ml final concentration) and PMSF (#8553) as protease inhibitors. A higher protein load for whole tissue extracts may be necessary when only a small portion of the cells in the tissue includes the post-translationally modified target. However, it is often necessary to increase the total protein load to at least 100 ug per lane for detection of modified targets (e.g., phosphorylated and cleaved) in whole tissue extracts. A list of recommended controls for many of our antibodies can be found on our Control Treatments by Target table.Ī protein load of at least 20-30 ug per lane is recommended for whole cell extracts and for detection of total/unmodified targets in whole tissue extracts. We always recommend including a known positive control to confirm experimental results. We recommend using expression profiling tools such as BioGPS or The Human Protein Atlas as well as scientific literature to check whether or not your cells or animal tissues are expected to sufficiently express the target protein of interest. Low Protein Expression in Tissue or Cell Line We recommend always using freshly diluted antibody for optimal results. Reusing diluted antibody is not recommended because the antibody is less stable after dilution and older dilution buffer is prone to microbial or fungal contamination. Select a troubleshooting topic of interest: To learn more about planning your western blot experiments, check out our Western Blotting Experimental Guidelines. A well-planned experiment, with appropriate controls, treatments, and conditions, is often the first step toward obtaining improved results. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |