I am trying to extract a soluble protein from Insect cells. After infection I see cell swelling. I harvest the cells at about 45-50% cell viability (3-4 days post infection). I see cell swelling. I have confirmed the bacmid prior to transfection with PCR.
When I harvest the cells and do lysis, I see all the protein of interest in the cell debris. I have tried 0.1% Triton X100 and NP40 separately in lysis buffer. My lysis buffer is 20mM Tris, 300mM NaCl, 0.05mM EDTA, 0.1% Triton X100 or NP40. I do not know what is happening. Is there something I am doing wrong? What could I do more to extract my POI from the cell debris? Any suggestion would be really helpful.
As sebastian suggested most likely you have problem with lysis. Why are you collecting cells after 3-4 days of infecting - do you really need such time of incubation - by cutting that time you can easily do 2 preps instead of one? Did you optimized your titer so that you can collect after 48 hrs ? One more suggestion is you can also increase percentage of triton / np-40 and your buffer dosent include protease inhibitors
Thanks for the suggestion.
Mahesh: I add PMSF in my lysis buffer.
I will try harvesting the cells earlier but at 48 hours there seems to be very little visible swelling under microscope, its not quantitative though. But I will try harvesting them earlier.
Sebastian: I shifted to insect cells a sin e.coli I was getting all the protein in the cell debris as well. I had tried different induction temps and different IPTG concentrations but didnt help.
Any more suggestions would help a lot. I am thinking may be my POI is forming inclusion bodies or aggregates of some sort. So i was looking into inclusion body solubilization techniques. Do you think that it will help?
For insect cells- i will use 1mM edta, at least 1X PI, 1mM pmsf, and will check if higher percentage of NP-40 helps. Swelling is not the stand alone criteria, you can do 2 ml cultures , harvest at different times and check yield
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