antibody purification from serum

[Dr.ThomDob]

hello everyone,

 

I would like to quantify my favorite protein from plant among different samples and the best strategy for me is to go to western blotting. Unfortunately, no antibody is now avaibable neither commercially nor in a home-made to target this protein. However, a company offered me the possibility to use an antiserum they produced in rabbit against my favorite protein.

I will first check with the unpurified serum its specifity (I have some under- and overexpressing lines that I can use as control) but in the worse case (and the most probable), this serum will not be specific enough.

My idea is then to purify the antibodies targetting my protein epitopes. To do so, I planned to produce my protein in bacteria with a tag, to purify it with the tag and then to use this purified protein as a bait to do an affinity chromatography on my serum.

 

I would like to know, first, if it makes sense to you as a strategy?

Then, if you have any robust method to suggest me for protein production (arabinose or IPTG), a bacteria strain to suggest (BL21Ai, BL21 Rosetta, ...) and also if you see any tricky thing I will have to consider before starting?

Finally, if anyone of you already experienced the serum purification and can give me some powerful advice and suggestions?

 

I thank all of you in advance, at least for reading .

 

Tom