CST, 7018S, PathScan® Sandwich ELISA Lysis Buffer (1X)

Buffer for studying in the research area. Product Usage Information 1. Thaw 10x buffer at 24-30°C, mixing end-over-end. 2. Dilute 10X ELISA Lysis Buffer to a 1X solution using ddH2O. 3. Chill 1X buffer on ice and add 1mM PMSF just prior to use. Lysis: All reagents and lysates must be kept cold. 1. Treat cells as desired. 2. Wash plate with PBS to remove residual media. 3. Add 1X to cells: (a) For adherent cells, add 500-1000 µl of 1X ELISA Lysis buffer/10 cm dish. (b) For non-adherent cells, judgment must be used as to amount of lysis buffer to add. Addition of volume equal to cell pellet is generally appropriate 4. Incubate plate on ice for 2 minutes. 5. Collect cell lysate solution for use. 6. Centrifuge extract for 5 minutes at 14,000 x g in a cold microfuge. 7. Remove supernatant for use. Additional protease inhibitors can be added to the 1X lysis buffer without any difficulties. Storage 1X ELISA Lysis Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 20 mM sodium Pyrophosphate, 25 mM Sodium Fluoride, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin For long term storage, keep 10X buffer at -20°C If buffer will be continually used, it is recommended that the 10X buffer be kept at 4°C for 1-2 weeks. Aliquoting of 10X buffer is recommended if many small experiments are to be performed. 1X solution can also be aliquotted into single use aliquots and stored at -20°C. Background PathScan Sandwich ELISA Lysis buffer (1X) was developed for ELISA applications and is formulated to better inhibit Serine / Threonine class of phosphatases after cell extracts are made.