Following induction for expression of the recombinant protein, E. coli culture was pelleted by centrifugation and resuspended in lysis buffer containing protease inhibitors. The cells were lysed using enzymatic lysis and sonication. The insoluble fraction of the lysate was subsequently separated from the soluble fraction using centrifugation and a sample of the soluble fraction was used to determine protein concentration by Lowry assay with a commercially available kit. The protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.
Size
200 μg
Concentration
1.0 mg/mL
Applications
Western Blot. Ready-to-use lysates are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Lysates are prepared in denaturing buffer WITHOUT dissociating agents (i.e. no 2-mercaptoethanol or dithiothreitol has been added). If dissociating conditions are desired, add reducing agent prior to heating. Gently invert vial several times to ensure that the solution is homogeneous and that any precipitated material is re-dissolved. A loading volume of 0.25 to 2.0 μL is recommended for SDS-PAGE. The product is supplied in 1X loading buffer and is ready-to-use.