I want to extract the target protein from insect cells. In a typical purification, starting from a pellet of insect cells culture, I suspend the cells in lysis buffer (50 mM TRIS pH8.5, 100 mM NaCl, 1% tween20, 1 mM PMSF, 5mM 2-mercaptoethanol and kept on shaking at 4 ⁰C. after that mix was spun down for 15 min at 10000rpm to remove insoluble particles. I am not able to purify my target protein. Can anyone suggest how to purify target protein from SF9 cells transfected with baculovirus?
Dear Shetaal
1- Could you tell me more detection method of your target protein?
2- Do you need to purify your Rec.protein from "Transfected " or 'Infected" cells , since the amount of protein expression in transfected cells is very low ??
3- what about the CPE and after how many hours do you collect your cells ?
4- Are you use a protein tag (like 6 his tag) or not ?!!
Best Regards
Hello Shetaal,
do you use a dounce homogenizer or something else (Sonic) to disrupt your cells? You are using a buffer system that has "physiologic" salt concentrations.
I wash my SF9 cells with a hypotonic buffer, disrupt them with dounce homogeniser and then I centrifuge them again.
hello ulrike
hello mohamed
i want to purify my recombinat protein from transfected cells and i am using the ^ His tag protein.
Hi Shetaal , the transfected cells yield a very low conc. of recombinant baculoviruses which may be the cause why you can't purify your target protein.
My recommendation is to infect new SF-9 cells with the transfection supernatant to amplify the recombinant virus and do another trial , you can check my paper for further details.
Wish you all luck !
hi mohamed,
i go through your research article . i want to ask you , have you purified your recombinant protein from transfected cells? your expression is very good. but about purification, there is nothing.
Dear Sheetal , Actually i don't purify my target protein , i have collected the transfected cells , prepared the virus stocks and infect new cells , the recombinant protein is detected directly by western blot and other methods without purification.
1. Harvest the infected SF9 insect cells by centrifuging the cell culture at low speed (500-2,000 x g). 2. Resuspend the pellet in lysis buffer containing an appropriate protease inhibitor cocktail. 3. Lyse the cells by sonication or by passing them through a French pressure cell. 4. Centrifuge the lysate at high speed (16,000 x g) to remove cell debris and unlysed cells. 5. If necessary, apply the lysate to a column containing a specific affinity ligand that binds to the target protein. 6. Elute the target protein from the column using an appropriate elution buffer. 7. Analyze the eluate for the presence of the target protein by SDS-PAGE, Western blotting, or other methods. 8. Purify the target protein further by chromatography (e.g., gel filtration or ion-exchange chromatography) or by affinity chromatography if needed.
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