how to confirm expression - (Dec/28/2008 )
I currently reach a bottle neck on my work.
We would like to identify the expression of alternative splicing variants of our target gene.
Other cell lines I tested either there was no expression or relatively strong expression; however, 2 cell lines, PC-28 and MCF, show something interesting.
This post will focus on MCF (gel of PCR result shown in PCR-2 and L3.6 is used as positive control).
For all primer set appeared, F873+R1584 seems to have highest efficiency compare to other primer set under same condition (tested by gradient PCR).
As shown in gel image, MCF has very low or basically no expression of our target gene. However, western blot result show relative strong protein product (identified by the size of protein band) in MCF cell line.
In the attached image, using primer F1791+R2720 and MCF treated with NPCD (5 uM for 24 hr and 2.5 uM for the next 48 hr), PCR produces 2 identifiable bands (pointed by arrows).
These 2 bands were sent for sequencing. Interestingly, the upper band (band A) is our target gene; however, only 350 bp at 3’ end sequence match our target gene and for the part match, only 90% homologous to the data base of our target gene.
My task is to make conclusion: do MCF call express our target gene?
Is band A I sequenced truly our target gene?
If MCF cell do express the RNA of our target gene, why only 1 primer set can identify it, but not the best efficient set.
If MCF cell do not express our target gene, what produce this positive sequencing result?
What we detect in Western Blot?
Or is there any way we can prolong protein half life so much, so that consistent expression of mRNA is not necessary?
Can anyone suggest other methods to test my task?
What antibody do you use for the WB? Monoclonal or polyclonal? And where does it bind your protein? Maybe it just binds the homologous region?
Did your database search suggest you any other gene than yours?
Did your database search suggest you any other gene than yours?
Thank you response
I think they have try 4 antibodies that contain both monoclonal and polyclonal, and recognizing different location of protein. Because of disagreement between RNA and protein results, they use couple antibodies to ensure protein expression result.
Although data did not fully agree with each other, all 4 antibodies detect some level of protein.
Although our target gene belong to a 4 members family, the sequencing result (only 90 % homologous and 3/4 shorter) only give 1 hit and that is our target gene.