Screening experiments of descaling agents for solubilizing membrane proteins
Screening experiments of descaling agents for solubilizing membrane proteins
Although TriumX-100 is a good descaling agent for solubilizing insulin receptors from murine liver plasma membranes, it may not be suitable for solubilization of other membrane proteins. Here, a strategy for screening a descaling agent to determine whether it is suitable for solubilizing a particular membrane protein to be isolated is presented. This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.
Operation method
Screening experiments of descaling agents for solubilizing membrane proteins Move Procedure For more product details, please visit Aladdin Scientific website.
1) Suspend the membrane preparation in the buffer system of choice (e.g., phosphate buffer or HEPES buffer). The buffer concentration should be 50 mmol/L and the pH should be close to the pKa of the buffer. The final protein concentration should be approximately 10 mg/ml. Store this membrane suspension at 4°C.
2) Prepare a 10% (100 mg/ml) reservoir of each descaler of interest, and dilute each descaler reservoir to 0.2%, 1%, 2%, and 6% with buffer.
Note: 1. Trichosanthin can only be solubilized to 4%. 2. A descaler kit is available from Boehringer Mannheim, which includes samples of nine non-ionic and three partially ionic descalers commonly used to analyze and purify membrane proteins.
3) Aliquots of the preparative descaler solution (from step 2) are mixed with an aliquot of the membrane preparation (from step 1). In each tube, the final concentration of membrane protein is 5 mg/ml and the final concentration of descaling agent is 0.1%, 0.5%, 1%, 3%, and 5%, respectively. Another sample was prepared and diluted with buffer without descaling agent. This gives a series of 0, 0.2, 1, 2, 6 and 10 descaler to protein ratios (i.e. mg descaler/mg protein) in the test tube.
4) Mix the mixture for 1 h at 4°C with stirring.
NOTE: Foaming should be avoided as it will denature the proteins.
5) Centrifuge the mixture at 100,000 g for 1 h at 4°C.
6) Transfer the supernatant to a clean test tube. Resuspend each precipitate in an equal volume of buffer.
7) Determine the protein activity of each supernatant and precipitate separately.
Note: Compare the protein activity in each supernatant and precipitate to see if the descaling agent solubilizes proteins from the membrane as the concentration of the descaling agent increases. The protein activity should be plotted against the descaler-protein ratio.