I have a stx2 236 TALE-A protein in glycerol stock at -20C. It's concentration is 3.2uM. I did buffer exchange with HEPES buffer 3 times (100uL protein+ 300uL of buffer each time) in a micro centrifuge at 10,000g for 5 min. I collected the flow-through each time. I recovered the protein at 1,000g for 2 min. I did Bradford assay for the recovered protein and flow-through. The protein concentration was determined to be 1.56uM and the flow-throughs had no protein in them. Why did I observe this loss from 3.2uM to 1.56uM?
Hi!
You can try using some glycerol (2,5-5%) in your HEPES buffer. This can avoid protein sticking to the filters.
Also, adjust salt concentration to avoid protein agregation and clogging.
Wash the filter with some buffer to increase recovery.
Best
Jacó
It sounds like your protein stuck to the ultrafiltration cartridge, possibly due to local precipitation. I usually rinse the ultrafiltration membrane a couple of times with a small volume of fresh buffer in order to recover material that may stick to it.
Greetings,
I guess you are using Amicon Centricons to exhange the buffer, am I right? I don't know if you miss the filter equilibration step when typing your answer, or when performing centrifugation. Anyway, prior to put your protein in the Amicon cartridge, you have to perform a centrifugation run using only fresh buffer, 5 mL should be enough. This is needed so the proteins contained in your sample don't denature when passing through the membrane. I'm almost sure that your main sample took a cloudy appearence once the centrifugation run came to an end. Furthermore, if this doesn't solve your problem, you can always use diafiltration membrane to perform dialysis against 300 - 500 mL of your desired buffer, Millipore handles a 6 kDa membrane that gives pretty good results when the dialysis protocol is performed over < 4°C and continuous agitation.
Good luck
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