Hello everyone,
I'm here to seek suggestions for fixing of yeast cells with formaldehyde, which are later to be visualized under microscope for tracking of a membrane protein tagged with GFP.
I fixed the cells with 4% formaldehyde and found GFP fluorescence getting quenched completely. So I tried 1% formaldehyde for 10 mins and found the fluorescence to be retained. Good news!! Yet I began to doubt whether formaldehyde has worked or not (cross linked the proteins or not). So I did a SDS PAGE of unfixed and fixed cells, in an expectation that fixed cells would show shift of proteins towards higher molecular weight (cross linking of proteins sums up the weight). Shockingly, the stained bands of unfixed and fixed (5min and 10 min) were exactly the same. I have attached an image below.
My questions is,
How to know whether formaldehyde has fixed the cells?
That is how you know. It's a good experiment and it tells you that the efficacy of your formaldehyde treatment was minimal. You do see some increase at the very high mw end, but most of your proteins clearly aren't affected.
What you have there can be good enough for many experiments, but if you really have to achieve anything worthy of the word "fixation", you need to make the formalin fresh from solid PFA, with slow dissolution, boiling and all. It's a headache, but it's the only way to do it. You can't just buy ready-made formalin in a bottle (even though many vendors will be happy to sell it to you, with no mention of any problems!). The problem is, it loses potency rapidly.
John Schloendorn Thanks for your reply. I later treated mid log cells with 1%PFD for 10 mins and reinoculated them in fresh medium for growth at 30. I had control cells treated with phosphate buffer. After 7 hours of inoculation, control cells grew but not PFD treated cells. This was another way of testing whether 1%PFD could fix cells or not. But after 24 hours the control reached OD of 6.8 while PFD treated one touched 2.8. I suspect sluggish growth for PFD treated cells. If I wish to stop that growth as well, would increase the PFD treatment to 20 min or more.
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