I have cloned my protein into mammalian expression vector pVAC1 and subsequently transfected 6 ug of DNA into 293 cells using lipofectamine...protein will be secreted out of the cell, since vector has secretory signal..
how can I confirm the expressed protein?
I have tried with GFP C-terminal fusion even though sequence was correct I could not get the GFP expression.
I collected supernatant from transfected wells 3 wells (6 well plate) then concentrated it and in SDS-PAGE there are no visible bands..
the cell lysate was prepared using NP-40 lysis buffer, and even with that I did not get any visible bands..
What could be the reason.. how can I confirm the expression of protein apart from radio isotope, in vitro expression system?
Dear Appavoo,
Have you checked your transfection efficiency? Also you may try to use flag tag rather than GFP to detect your protein with western blotting if you do not have a specific antibody for your protein.
I hope that would help
Good luck
Dhamad sir,
transfection efficiency very good..i dont know about FLAG tag..can u brief, how to add it to the protein..
Try to increase the protein concentration by sonication for 5-10 sec thrice after lysis or supernatant collection...
Do you see a GFP signal down the microscope after 24, 48 and 72 hours of transfection? If the protein is to be secreted, you should still be able to see some signal intracellularly. If you do not see a GFP signal at these time points, I would sequence the plasmid (to validate the sequence and make sure the promoter is in place, and make sure there are no mutations in the cloned sequence) and maybe clone another protein in to see if that can be expressed using the same vector.
I would also co-transfect the cells with an RFP plasmid just to make sure the are transfected and to get a feel for the efficiency.
Hope that helps
Osama
Dear Appavoo,
The FLAG-tag is a polypeptide protein tag that can be added to a protein using recombinant DNA technology.The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da). Simply, you can integrate the FLAG-tag with your protein as you did with GFP. Then you can use anti- flag antibody to detect your protein.
Since your protein is secreted in the medium, you can do ELISA with proper controls (vector transfected cells- culture medium as negative control). Use RT-PCR to check atleast if mRNA is made.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques