3 Matching Annotations
  1. May 2019
    1. (ii) Stacking gel buffer: 1.0 M Tris-Cl pH 6.8 (iii) Resolving gel buffer: 1.5 M Tris-Cl pH 8.8 (iv) SDS stock: 10% (w/v) solution (v) Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh (vi) Gel running buffer (1X) (vii) Loading dye (6X): (viii) Lysis buffer (RIPA) Gels of 1.5 mm thickness were cast in the Biorad small gel apparatus. Resolving gel of 10% (10 ml) was made by mixing 4.2 ml 10% acrylamide, 3.1 ml water, 2.5 ml of 1.5 M Tris-Cl pH 8.8 and 0.1 ml of 10% SDS. Stacking gel (2 ml) was made by mixing 0.33 ml of 30% acrylamide, 1.4 ml of water, 0.25 ml of 1 M Tris-Cl pH 6.8 and 0.02 ml of 10% SDS. Gels were polymerized by the addition of TEMED (N,N,N′, N′-tetramethyl ethylene diamine) and APS (1/100th volume of gel mix). Sample preparation for gel loading was done as follows. Mid log and late log phase 10 ml cultures were centrifuged at 26000g and the cell pellet was resuspended in 0.5 ml RIPA buffer. Cells were sonicated on ice for 1 min at output power of 5 to get a cleared lysate. The culture lysate was centrifuged at 26000g to recover the clear supernatant. Total cell protein was quantified in the lysates using BCA kit reagents (BioRad) using the manufacturers protocol. Appropriate volume of cell lysate was mixed with the loading dye in a final concentration of 1X and loaded onto the gel. The gel was run at constant voltage of 60 V for stacking and 80 V for resolving gel
    2. The method followed was as described in Sambrook and Russell (2001). The following solutions were used to cast and run SDS-PAGE gels. (i) Acrylamide stock: 29% (w/v) acrylamide and 1% N,N′-methylene bisacrylamide
    3. Sodium dodecyl sulphate-polyacrlyamide gel electrophoresis (SDS-PAGE)