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Reflections on the use of protease inhibitor cocktails for preserving protein in tissue lysates
Author(s) -
Hoegler Carl S,
MurphyGoldberg Gabriela,
Ponessi Louis,
BlandoHoegler Charlene F
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.574.10
Subject(s) - protease , protease inhibitor (pharmacology) , western blot , trypsin , myosin , polyacrylamide gel electrophoresis , microbiology and biotechnology , pi , chemistry , gel electrophoresis , enzyme , trypsin inhibitor , biochemistry , biology , virus , virology , gene , antiretroviral therapy , viral load
Commercially‐available protease inhibitor cocktails (PI) are routinely included in tissue lysates to insure that there is no loss of protein integrity of tissue samples. In view of the paucity of published research, our study investigated whether such inclusion was efficacious. We examined the effects of four protease inhibitor cocktails on rat skeletal muscle protein lysates. Total protein band analysis was performed after polyacrylamide gel electrophoresis (PAGE); in addition, myosin light chain (MLC) banding was compared after Western blot. Our results showed that, although most of the tested PI's do not appear to alter the protein content or band pattern (from 10 to 250kD) of the samples, anomalies were evident. One PI significantly reduced the measured amount of MLC. In another experiment, muscle (biceps femoris) lysates were treated with protease (trypsin) and incubated at 37°C for one hour. The enzyme treatment eliminated all of the protein bands (including MLC). In trypsin‐treated lysates containing PI's, most protein bands were rescued from hydrolysis to varying degrees. Restoration of MLC bands varied with the PI. Our conclusion from these studies is precautionary; we recommend that investigators reflect on their routine use of any PI. (Support from SURE Program at MSMC and Pace Scholarly Research)