Comparison of Two Fluorescence Based Assays to a Sensitive SDS‐PAGE Method for Detection of Trace Proteases in Bovine Serum Albumin

Bovine serum albumin (BSA) is used in immunoassays both as a stabilizing protein and to block non‐specific binding. Proteases are prevalent throughout nature and as such native blocking proteins can contain varying levels of protease. Additionally, as immunoassays have become increasingly sensitive in detecting lower concentrations of analyte, blocking proteins that contained once acceptable low amounts of protease are now problematic. Many commercial BSA preparations contain low levels of proteases that have undesirable affects on immunoassays such as decreased activity, loss of stability and irreproducible results. We optimized and evaluated two commercially available protease detection kits which use modified casein as substrate. The casein is hydrolyzed by proteases in the sample resulting in an increase of fluorescence which is then measured in a microtiter format. The results were compared to protease levels determined by a very sensitive but time consuming SDS‐PAGE method, which detects protease induced degradation of casein in a BSA sample.