I generated different constructs for production of virus-like particle vaccine candidates using 293TT cells. Is there anyone having good experience in using 293TT cells for expression of recombinant proteins? I will be very glad if you are able to share your protocols or published papers for transfection, protein expression, cell lysis buffers and protein purification procedures. Many thanks for your help in advance.
Hi Alemu,
For the transfection of the expression vectors into HEK293 cells I have successfully used the PEI method. You can start with the web site of the Cold Spring Harbour Protocols http://cshprotocols.cshlp.org/content/2008/3/pdb.prot4978.full
I have used the ratio of DNA:PEI 1:4 (w:w). It is important to mix DNA with PEI using small volume (typically 10% of the total medium volume) of antibiotic/serum free DMEM first. After 10 min. of incubation, the rest of DMEM with antibiotic/serum should be added and the transfection mixture immediately loaded into the well or plate with the cells (at 70% confluency). Of course, the old medium should be removed first. For the cell lysis I recommend the buffers with 0,1% NP40, unless your protein doesn’t like this detergent.
Bone chance,
Ela
Dear Ela,
Many thanks for your answer and for sharing the protocol. I appreciate it!
- Badges
- Science method