I am currently trying to stain for PI3KBeta in MDA-MB-231 cells. My current issue is that 1) beta stains all over the cell and 2) there is an increase in signal at the edge of the cell and this increase in signal co-localizes with cellular ruffles (imaged in phase). My goal is to look at PI3Kbeta's recruitment to the plasma membrane in response to growth factor stimulation; however, an increase in staining at the edge of the cell that may be due to ruffling makes it very difficult to study this. Does anyone have any tips/experience/references that can help me analyze protein recruitment to the edge of the cell and reduce the effects of signal increase due to ruffling. Are there any techniques to keep the cell flat on the coverslip? thank you so much for your help!
I'm taking a guess, possibly if you have a positive control for membrane expression (transmembrane protein), say in Green color, and PI3Kbeta, Red color. Process growth factor (- neg cells, possibly serum starved cells) vs growth factor (+, pos cells). Fix with formaldehyde, permeabilize cells, stain and compare Red fluorescence/green fluorescence in - vs + cells. It should localize to the ruffles as you found. Excess ratio in +cells over -cells should give you the signal you're looking for. Image with confocal microscopy. Ruffles are a blessing in disguise.
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