Hi researchers,
I want to over express a protein in Hep3B cell line. The expressing vector I use is attached here. The packaging vectors are pMD2.G, pRSV-Rev and pMDLg/pRRE. The amount I use for each plasmid is also attached.
I use TransIT-2020 at a ratio 3:1 to transfect 239T cells (50% confluent in T75).
72h later I collect the supernatant, spin and filter through 0.45um.
The amount of filtered medium is around 10ml. Then I add 2ml fresh medium to make it 12ml in total and add protamine at final concentration 10ug/ml.
Next I put 3ml of this viruus containing medium to each well of 6-well plate, in which the Hep3B is at 50% confluent seeded one day before.
After one day I start hygromycin selection with a final concentration of 100ug/ml.
My problem is, the Hep3B can grow very well after one week selection but when I do western blot to check this protein, I cannot detect anything. The antibody I use is working well and at mRNA level, I can detect the increase. This problem also happend when I used another lentivirus system (1st gen). I really don't know what is happening.
Thanks for any advice to make it work!
Effect of misfolding. See the discussion of similar problem here: https://www.researchgate.net/post/Why_am_I_not_able_to_detect_6_HIS_tag
Thanks Andery! But I think this is not my problem. My colleague use the same vector and can detect the protein in another cell line. But he did several construct, only some of them can be detected. Maybe we had the same problem but don't know what it is.
If you haven't already, I would highly recommend you check the mRNA levels of your protein in the transduced cell line to at least confirm if the protein is transcribed in your particular cell line.
Also, try simply transfecting 293T cells with the vector expressing your protein and do an immunoblot for your protein just to see if that particular vector your have is working correctly. But if your colleague has successfully used that same vector then It may not be the problem.
You may also want to switch out the types of tags (His, Myc) on the protein or even move the tags from N to C terminal.
Did you reconstitute the gene by PCR? Did you sequenced your construct? May be you introduced some mutation for stop codons. I suggest to use the Retro-X™ Concentrator if the production/concentrations of your virus is low
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