Hello I am having a bit of an issue with my sf9 cells and trying to express the human PTH receptors. So:
I transform DH10 cells which give me blue/white colonies, which I also do a colony PCR to see if my insert is there and it is. I then proceed to do a bacmid purification (precipitate over night in isopropanol and 70% ethanol just before transfection) and transfect my cells on a 6 well plate. After 72 hours I harvest the media,replace it with 3ml new media and store the baculovirus in the fridge. 72 hours from that I do a western blot which shows my protein was there.
I infected 25ml of sf9 cells with 3ml of my virus on saturday at a density of 1x10(6) they had stopped dividing on monday (48 hours) and then today (72hours) I was going to harvest the virus. However my cells look incredibly stressed with lots of debris, but the media they are in looks perfectly normal, after centrifugation the pellet is normal coloured etc etc. I see no signs of contamination.
Even on the previous 6 well plate my cells looked incredibly stressed with lots of debris, but the media control was normal and insect control looked perfectly fine. I'm assuming I have a contamination that has led to this so I have filtered the virus and will run some more tests but any ideas on what is happening? I'm quite new to insect culture but assumed contaminations would be very apparent?
Thank you very much for reading!
Hi, I am also exploring the GPCR expression in Sf9 cells. However, after harvesting the cells, I did not observe any protein expression using the WB. I am wondering which type of buffer you used or is there any matters need attention. Thank you!
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