Testing expression of OVA gene - (Sep/27/2006 )
Hi
I am trying to express OVA gene in B16 cell line (Mouse). I tried polyclonal Rabbit anti OVA Antibody (FITC Labelled) to detect OVA protein in B 16 through Flow cytometry (Binding assay and sort) but could not get desired result. I suspect it has got a high level of non specific interaction. I used rabbit serum, changed binding assay conditions and even blocked the Anti OVA Ab with Ovalbumin followed by adding the B16 cells. But nothing worked. Untransfected B16 gives almost the same signal that the transfected one gives. Should I try monoclonal Ab? Has anyone used any specific commercial Ab for this purpose? Is there any other method to test the protein?
I have read somewhere that a Japanese group actually measured the OVA specific mRNA in the cell line by RT PCR for quantification? Is n’t that a too cumbersome way?
Cheers
Faham
I am trying to express OVA gene in B16 cell line (Mouse). I tried polyclonal Rabbit anti OVA Antibody (FITC Labelled) to detect OVA protein in B 16 through Flow cytometry (Binding assay and sort) but could not get desired result. I suspect it has got a high level of non specific interaction. I used rabbit serum, changed binding assay conditions and even blocked the Anti OVA Ab with Ovalbumin followed by adding the B16 cells. But nothing worked. Untransfected B16 gives almost the same signal that the transfected one gives. Should I try monoclonal Ab? Has anyone used any specific commercial Ab for this purpose? Is there any other method to test the protein?
I have read somewhere that a Japanese group actually measured the OVA specific mRNA in the cell line by RT PCR for quantification? Is n’t that a too cumbersome way?
Cheers
Faham
I dont know if this is off the topic: OVA is not a membrane protein unless you have a signal peptide and transmembrane sequences fused to the cDNA sequence in the plasmid construct. Maybe you need to permeate cell membrane then stain for it if it is a cytosolic protein?