hello friends
I have recently started with Insect cell culture work. I transfected SF9 cells in 6 well plate with Bacmid containing my GOI.
Even though in literature its metnioned to use P2 virus transfected cells for confirmation of protein expression.
Can i use the cells after 72 hrs of virus release to detect whether my GOI is expressing or not using His Ab in Western blotting.
Hi there,
Well in theory you can (and I have checked my P1 by WB). Problem is that will depend on the plasmid and level of expression of your GOI (and how good your Ab is). This is what I do:
1. Add 2 mL of cold PBS to the cells, scrape and transfer to a 15 mL falcon tube.
2. Centrifuge for 5 min at 3000 rpm (4°C). Remove supernatant.
3. Re-suspend each pellet in 500 μL 4 x denaturing protein sample buffer and transfer to 1.5 mL Eppendorf tube.
4. Boil 5 min.
5. Shear DNA/RNA by passing through 18G needle 8-10x.
6. Analyse 10 μL by western blotting to make sure that your protein is being expressed and which clone gives optimal expression.
Give it a try,
HTH
paolo
Hello Paolo
Thank you so much for simplifying it along with providing a protocol.
Gratitude
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