Antigen isolation

[Katiejo404]

I'm trying to strip the proteins from the surface of [I]Lactococcus lactis[I] to restimulate T cells after I've isolated them from a mouse previously injected with L. lactis. I've done the extraction with LiCl before, but we're trying a new approach with lysozyme right now and I'm not sure if all the steps are necessary. I incubated the bacterial cells with lysozyme in a 50mM Tris-HCl + 0.1M NaCl buffer at pH 7.8, centrifuged it to remove the cells, and then transferred the supernatant into dialysis bags. Currently, I am in the process of concentrating them by coating the bags with Sephadex, and once down to a small enough volume, I am supposed to dialyze this in the same buffer as used in the extraction, but I think that it would just take up a lot more water into the bags that would need to be removed again to reconcentrate the proteins. How necessary is it to do this dialysis? I recognize that the Tris-HCl and NaCl concentrations will increase with this method of concentration, but I am using such a small volume in the cultures that I'm not sure if the dialysis to reduce the Tris-HCl and NaCl concentrations is necessary. Any comments would be appreciated.
Also, if anyone has any better ideas on how to concentrate the proteins faster, that would be much appreciated. We are a fairly new research lab and not terribly well-funded, so we don't have very much higher end equipment, like an ultracentrifuge, but there has to be an easier way than coating 9 dialysis bags with Sephadex 4 times a day for 4 days to remove ~10mL of water.

Thanks so much,
Katie

[Katiejo404]

Alright, I'm going to simplify my question. Has anybody used a drying agent other than Sephadex (G-10) to remove water from a solution by osmosis through a dialysis bag? The Sephadex is working to remove about 0.5-1mL per treatment and I need to remove about 10-15mL total, so this takes a while. If anyone knows of a drying agent that can remove the water in greater volumes, or doesn't need to be changed as often, I would love to hear.

Thanks,
Katie

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