I have to generate antibodies (Ab) specific for one isoform of insect cytochrome P450 for studying expression of this isoform through western blotting and ELISA. In this regard, I have tried to express this gene in bacteria (M15 strain) using pQE30Xa vector, but I could not able to achieve. Therefore, I am looking for alternative methods then I heard of chemically synthesized peptides (8-10 amino acids in length) are also used in ‘Ab’ generation. However, I have some queries regarding specificity of this Ab, whether this Ab is specifically hybridized only to respective isoform or generally it binds to different isoforms of cytochrome P450. Some of the studies have shown that, Ab generated from truncated proteins resulted in non-specific hybridization. In view of that, how about specificity of Ab generated from chemically synthesized small peptide molecules (8-10 amino acids in length). Please suggest me whether shall I go ahead with this or any alternatives are there?
The quality of polyclonal antibodies generated against synthetic peptides is highly variable. Many commercial antibodies generated in this manner are almost completely worthless, while others work quite well. Unfortunately, you won't know until you try. Sometimes polyclonal antisera with a lot of nonspecific binding can be improved by affinity purification of antibodies using your target protein. With regards to isoform specificity, you should try to choose a peptide which is specific to your particular isoform of interest, and not overly similar to anything else.
Finally, a note about protein expression - have you tried expressing it in insect cells? Many proteins which cannot be expressed in bacteria work fine in insect cells, and making baculovirus is not difficult.
Short synthetic peptides for immunization is a great strategy. You can pick a peptide with good hydrophilicity and that is unique by protein BLAST, order the synthetic peptide, KLH couple it, and send it to a company like Covance for immunization and bleed collection. Including a Cys residue on the N-terminus of your peptide is good, because then you can use Sulfo-link columns for affinity purification after you protein A purify the immunoglobulin. There are some companies with a nice offer for custom polyclonal antibody production, so you should probably just ask them for a quote so that you don't have to learn every step and buy everything if you are only going to do it once.
Just an idea, because I have never worked with Ab generated against synthetic peptides. If the sequences of all the isoforms are available then maybe you can perform alignment of the sequences to find a unique oligopeptide for the gene that your are working with. Then see if you can use that as the synthetic peptide. I stand corrected if my idea is not feasible.
- Badges
- Science topic