Quantitative Total Protein Estimation

Total protein standards were prepared by diluting 5 mg/mL bovine serum albumin (BSA) in DMD lysis buffer, and then diluting serially 1:2 six times (5–0.078 mg/mL). Test samples were thawed on ice. Aliquots were removed for total protein measurement. A total of 1 μL of each test sample or BSA standard was pipetted onto a nitrocellulose membrane, at least in duplicate measurement. After air drying, the membrane was incubated with 1X LI-COR REVERT^™ stain (LI-COR, Lincoln, NE) as per the vendor protocol. The membrane was incubated in the stain for 5 min, and then washed twice in equal volumes of 1X LI-COR REVERT^™ Wash Buffer. The dots on the membrane were visualized and quantitated on a LI-COR Odyssey CLX^™ Imaging System (LI-COR, Lincoln, NE). Total protein on the samples was determined by interpolation of dot-blot fluorescence intensities into the BSA standard curve as described previously (36 (link), 37 (link)).