Too cold for agarose beads to survive? - Antibody Affinity Purification (Mar/03/2009 )
I have a column for affinity purification of antibodies. The base resin is agarose to which a 19 aa peptide antigen was coupled to. The column was however inadvertedly placed into a minus 20 freezer for 6 weeks. I really don't want to risk my precious IgG prep on this column to test if it is still alive. Had anyone have such an experience and can you tell me what would happen to peptide-coupled agarose columns when they have been frozen-thawed? Thanks
what happens to the peptide upon freezing depends on the peptide.
the agarose is another story. agarose beads may be broken by ice crystals when frozen. this may not affect the binding ability but will certainly affect the flow rate of the column.
you may be able to use the packing for batch separations but i would be reluctant to use it in the column.
you can check it by running buffer through and see how the flow rate changes.
Wouldn't you have to re-pack the column anyway?
Why not just give the gel a few rinses in a test tube, take a small portion and react with your protein, wash, elute. If your bindable protein remains in the wash then the gel is fini. But, if the protein elutes then voila repack the column and go for it! (providing you have an acceptable flow rate.)
sgt4boston on Mar 4 2009, 12:50 PM said:
no. you can perform batch separations in the tube.
add the antibody and mix, spin down the resin, remove supernate, wash with buffer (mix, spin, remove supe) a couple of times, add elution buffer, mix, spin, collect supe, repeat elution.
no need to repack column.
Sorry I had a day off so I didn't reply immediately to say great thanks to you guys for being so kind to help.
I appreciate your sharing the knowledge about effects on the flow rate. And the batch separation is a smart idea indeed. However for the uncoupling of the purified antibody I would have to use acidic buffers. Neutralization in batch experiments seems to take longer time that may pose a risk to my Ab. I think I would have to produce a new column, but I found this forum so useful. Thankyou all again and have a great weekend.
bachai on Mar 6 2009, 02:09 AM said:
the time it would take to neutralize for batch processing would not be too long for the stability of your antibody.
when eluting from a column, some fractionate into the neutralization buffer, some add it after elution, similar result.
during batch elution you only wait for the time to spin and decant (or pipette) before you neutralize. it takes a similar amount of time as to column elute.