Purified G actin and got 42kda band and an additional band just below it. While performing G actin polymerization and labeling with rhodamine-phalloidin I didn't observe filaments under the fluorescence microscope
Hi Sneha,
Which cell type you used? I guess you checked in epifluorescence mode or confocal mode. Try in TIRF mode. It will show filaments at the footprint of the cell.
Ekta Mayur Dembla Thank you for your suggestion, I am isolating actin from chicken breast muscle and yes, I used epi-illumination mode but the filaments should be of a few microns in length so I cross-checked in bright-field with a 60x oil objective, and still couldn't see anything. I am attaching the gel and microscopy images along, what do you make of it? And thank you again for all your help.
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