Protein purification for rabbit injection - SDS-PAGE gel, excise band, inject into rabbit?? (Nov/29/2006 )
Hello immunology boffins,
I am purifying a protein for antibody production. It's not a cloned protein, there is no tag, and no way of getting a sufficient amount pure enough for rabbit injection without running an SDS-PAGE. 
What I want to know is: Can I run the gel, stain it with coomassie, chop out my band of interest, macerate it well (in PBS?) and inject it into a rabbit? I've heard that the acrylamide acts as an adjuvant (not surprising really!), and that you can get an anibody from this.
Has anyone tried this (with success or not)? Or is there a better way of getting my antigen?
Cheers, and have a nice day in the lab!
Nicole
It may work but I'm not sure about injecting dye with protein. I would rather run two lanes - one with enough protein to see stained band, other with much more protein. After run I would cut gel in two and stain first lane just to see where should I cut the other lane.
I don't want to play an expert whose I'm not but there's always method to purify protein. It's only question of avliable protein, time and equipment. Starting from basics - organic solvent precipitation (eg. acetone, ethanol), ammonium sulphate fractionation, gel filtration, ion exchange, affinity (eg. lectin) etc.
Hi there!
I'm trying to do a similar thing but i don't want to inject gel (spoke to a company, they said you get antibody against the gel and dye too) so people recommended electroelution of protein from gel. there are tube for one time use gebaflex
Other companies sell the same you can use it with agarose gel tank
you can elute clean protein from sds gels and it gets dialysed as well
Stardust
I am purifying a protein for antibody production. It's not a cloned protein, there is no tag, and no way of getting a sufficient amount pure enough for rabbit injection without running an SDS-PAGE.
What I want to know is: Can I run the gel, stain it with coomassie, chop out my band of interest, macerate it well (in PBS?) and inject it into a rabbit? I've heard that the acrylamide acts as an adjuvant (not surprising really!), and that you can get an anibody from this.
Has anyone tried this (with success or not)? Or is there a better way of getting my antigen?
Cheers, and have a nice day in the lab!
Nicole
years ago we did so as you suggest; some advices:
immunize several rabbits (at least 3 to 4)
try to electroelute your protein so that it is in solution and is better to inject; use other adjuvants than AA;
with polypeptides from gel you must be sure that it is pure, and if you will get an Ab it will be against denaturated protein which is preferably to use for Western blots
try to get a peptide with your epitope of interest, and bind it covalently to KHL or BSA, and try in parallel immunization with these constructs
I am purifying a protein for antibody production. It's not a cloned protein, there is no tag, and no way of getting a sufficient amount pure enough for rabbit injection without running an SDS-PAGE.
What I want to know is: Can I run the gel, stain it with coomassie, chop out my band of interest, macerate it well (in PBS?) and inject it into a rabbit? I've heard that the acrylamide acts as an adjuvant (not surprising really!), and that you can get an anibody from this.
Has anyone tried this (with success or not)? Or is there a better way of getting my antigen?
Cheers, and have a nice day in the lab!
Nicole
we've done it a few times. it works.
we have also injected into mice and made monoclonals.
can use zinc staining. It stains your gel's background and the process only took you like mins time.
Excise the gel and subjected to electroelute.