I am trying to prepare samples for IP. The pellet size is good and I am using 1% Tx-100 and using a syringe to further lyse the cells and maintain protein-protein interactions. I keep having a small amount of pellet remain in the solution and protein concentration ends up being too low for IP. No matter how much buffer I add or I pipette/syringe the mixture, the pellet does not disappear and I feel like I am losing protein concentration because of that. How can I increase my protein concentration?
• To increase the concentration of protein in a lysate, you can use several methods:
o Centrifugation: Use ultracentrifugation to remove cellular debris and concentrate the protein.
o Precipitation: Use a method like ammonium sulfate precipitation to concentrate proteins by precipitating them out of solution, followed by resuspension in a smaller volume.
o Dialysis: Dialyze against a solution with a lower volume or use lyophilization (freeze-drying) to reduce the volume of your lysate.
o Concentration Devices: Use centrifugal filter devices that allow you to concentrate your sample by removing the solvent while retaining the proteins.
o Buffer Exchange: If necessary, consider buffer exchange to remove salts or other contaminants that could interfere with downstream applications while concentrating the lysate.
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