5 replies to this topic

[zuda]

hello everyone
  i have  a question, i cloned a part of CDS of my gene of interest, and not complete CDS. In general the full cds codes for approximately 600 amino acids, but the region i cloned spans 111 amino acids. The question is, if i transfect my cells with the cloned CDS and over-express the gene of interest, will i be able to detect a protein of 66kda? or i will detect only 12-15kda. the base size of my protein is 66KDa. I am very worried. Please somebody suggest somethig.

[Curtis]

it will be the smaller size, but to be exactly sure about the weight you can copy and paste your protein sequence in this online software.


http://www.encorbio....ols/Prot-MW.htm

There are a lot of calculators online.

I just want to know , do you have start and stop codon in the shorter fragment? Are you doing SDS-PAGE? Western blot? because if you ware running western blot your antibody might not detect your expressed protein in shorter form.

[zuda]

Thanks for your reply, i checked and as expected i got the size 12 kda, but the strange thing is i got the band at 66 kda using the partial cds transfected cells. I am doing western blotting, does it means i am doing something wrong? I also saw some papers which have cloned partial CDS and then they performed western blotting to check the expression of their gene of interest.
Apart from western blotting, qrt-pcr results are also showing that upon transfection of partial cds, the expression of gene is induced.
Please suggest me something.

[bob1]

I would suspect that the 66 kDa band you are seeing is not your protein of interest if you have only transfected in a truncated version, rather it is likely to be non-specific binding of the antibody.

It could be that the cells you are using have this protein normally (depends on the cell line and the protein source species).

There is nothing wrong with doing western blots, they are an established and well used technique that most people accept as a valid experimental procedure.

[zuda]

thanks a lot for your replies, i really appreciate that, i guess i should reclone.

[Jason_LuYZ]

zuda, on 23 July 2012 - 05:26 AM, said:

Thanks for your reply, i checked and as expected i got the size 12 kda, but the strange thing is i got the band at 66 kda using the partial cds transfected cells. I am doing western blotting, does it means i am doing something wrong? I also saw some papers which have cloned partial CDS and then they performed western blotting to check the expression of their gene of interest.
Apart from western blotting, qrt-pcr results are also showing that upon transfection of partial cds, the expression of gene is induced.
Please suggest me something.

I think this is a normal thing. If the gene of interest endogeneously  expressed in your transfected cells, it obviously may detected expression due to the primary antibody you used. The key for this the antibody you used in this western blot. It seems not to distinguish these proteins.   However, you can used different antibodies that detected different immunogen epitope to furher identify your target protein(Truncated protein). Good luck