How to get rid of gelatin?
May 27, 2008 8:04 AM Subscribe
Biochemists, cell biologists and assorted biotech types, what's your favorite method for removing gelatin from antibody solutions? No amines allowed!
We have purchased a whole bunch of antibodies which contain 1 mg/ml gelatin as a stabilizer (rather than BSA) and we need to get rid of the gelatin before we attach the Abs to AminoLink resin, which reacts with primary amines.
This gelatin is an acid hydrolysate of bovine collagen and the molecular weights range from ~200 kD down to itty bitty. The pIs are all over the map too. The Abs are polyclonals from goat and rabbit and we have about 200 µg in each pricey little tube.
We've tried Protein G but find that the acidic elution conditions wipe out the Ab activity pretty badly. Obviously we haven't tried every eluant under the sun, so do you have any faves that preserve activity while giving good recovery? Remember, no amines allowed (we don't want to dialyze or desalt because we'll lose a good chunk of the Ab at this step).
Melon Gel (from Pierce) gets rid of about 2/3 of the gelatin but that's not good enough. 2 cycles of Melon Gel are no better than 1. Ion exchange on CM Sephadex in 10 mM NaPi at pH 6.5 also removes about 2/3 of the gelatin.
Any ideas? References would be greatly appreciated, too. Thanks in advance!
We have purchased a whole bunch of antibodies which contain 1 mg/ml gelatin as a stabilizer (rather than BSA) and we need to get rid of the gelatin before we attach the Abs to AminoLink resin, which reacts with primary amines.
This gelatin is an acid hydrolysate of bovine collagen and the molecular weights range from ~200 kD down to itty bitty. The pIs are all over the map too. The Abs are polyclonals from goat and rabbit and we have about 200 µg in each pricey little tube.
We've tried Protein G but find that the acidic elution conditions wipe out the Ab activity pretty badly. Obviously we haven't tried every eluant under the sun, so do you have any faves that preserve activity while giving good recovery? Remember, no amines allowed (we don't want to dialyze or desalt because we'll lose a good chunk of the Ab at this step).
Melon Gel (from Pierce) gets rid of about 2/3 of the gelatin but that's not good enough. 2 cycles of Melon Gel are no better than 1. Ion exchange on CM Sephadex in 10 mM NaPi at pH 6.5 also removes about 2/3 of the gelatin.
Any ideas? References would be greatly appreciated, too. Thanks in advance!
Response by poster: Thanks purplemonkie, do you have a reference or recipe for MgCl2 elution? I've never tried that.
We tried binding the partially-cleaned Abs to AminoLink and got a fair amount of protein bound to the resin, but the specific activity was dismal. Either the gelatin peptides out-competed the Ab, or the Melon Gel destroyed Ab activity. My hunch is that it's the former. We're going to look into this further, but I'm hoping somebody out there has already solved this problem!
posted by Quietgal at 10:28 AM on May 27, 2008
We tried binding the partially-cleaned Abs to AminoLink and got a fair amount of protein bound to the resin, but the specific activity was dismal. Either the gelatin peptides out-competed the Ab, or the Melon Gel destroyed Ab activity. My hunch is that it's the former. We're going to look into this further, but I'm hoping somebody out there has already solved this problem!
posted by Quietgal at 10:28 AM on May 27, 2008
Response by poster: Followup for the benefit of any biotechies who might have googled into this question:
Eh, we decided to ask the supplier (Santa Cruz Biotechnology) to do a custom order w/o gelatin for us. I mention it here because they're actually quite reasonable: the minimum order for this custom formulation is 2 vials but the price per vial is the same as the regular product. And they don't repurify the Ab over Protein G (which would expose the Ab to another harsh acid elution, reducing its activity); they go back to their frozen concentrated stock which has no gelatin and dilute it to normal strength.
We fooled around a little with various cleanup methods and decided that buying new, clean Ab was actually more cost-effective than reprocessing the stuff we already have.
I don't work for SCBt, I don't know anybody who works there, but I figure they deserve a little plug for being so helpful and economical.
posted by Quietgal at 10:24 AM on June 5, 2008
Eh, we decided to ask the supplier (Santa Cruz Biotechnology) to do a custom order w/o gelatin for us. I mention it here because they're actually quite reasonable: the minimum order for this custom formulation is 2 vials but the price per vial is the same as the regular product. And they don't repurify the Ab over Protein G (which would expose the Ab to another harsh acid elution, reducing its activity); they go back to their frozen concentrated stock which has no gelatin and dilute it to normal strength.
We fooled around a little with various cleanup methods and decided that buying new, clean Ab was actually more cost-effective than reprocessing the stuff we already have.
I don't work for SCBt, I don't know anybody who works there, but I figure they deserve a little plug for being so helpful and economical.
posted by Quietgal at 10:24 AM on June 5, 2008
This thread is closed to new comments.
(Incidentally, are you certain that removing 2/3 of the gelatin with Melon Gel is not enough? Back when I was dealing with this issue I spoke with a Pierce rep who told me that as long as the total concentration of amine in the binding solution doesn't exceed the capacity of the resin -- which is up to 20 mg/mL for AminoLink -- antibody binding should not be significantly affected. I suppose he could have been feeding me a line, but it's a thought!)
posted by purplemonkie at 9:11 AM on May 27, 2008