I am purifying a 35 kDa protein, its temperamental and oddly, it crashes in low imidazole rather than in a higher concentration of imidazole. Anyways, I wanted to dialyse it for SAXS analysis at an initial concentration of 5mg/ml. However, after dialysis, when I took it to SAXS, and measured the concentration, it was 0.5 mg/ml. I can't really compute as in to why this happened. Anyone with ideas? Is this normal?
Hey Liyana!
Well, I would guess that if you still have some air inside the dialysis tube, more liquid can be transferred into the tube during dialysis than there has been initially before. But a difference of factor 10 (as in your case) does not seem normal to me and I would also check the tube, as Gaurav suggested. Perhaps do a backup closure using plastic clamps behind the nodes just to be sure that nothing is escaping.
Cheers,
Michael
Hi!
Do you correctly tie your dialysis tube..normally this shouldn't happened.You can try to concentrate your protein in Vivaspin Centrifugal Concentrator. Choose the size according to your protein..thats better option for the protein concentration..and yes you tie the dialysis tube properly check with some buffer or milli-q before adding your protein sample..hope this can help you..gud luck
Hey Liyana!
Well, I would guess that if you still have some air inside the dialysis tube, more liquid can be transferred into the tube during dialysis than there has been initially before. But a difference of factor 10 (as in your case) does not seem normal to me and I would also check the tube, as Gaurav suggested. Perhaps do a backup closure using plastic clamps behind the nodes just to be sure that nothing is escaping.
Cheers,
Michael
Hello Liyana,
some proteins do not really stand dialysis and you may loose some, but such a difference is huge and suspect. I agree with the answers by Gaurav and Michael, do try to secure your dialysis tube when you tie it, use also some clamps. Or maybe you could use some dialysis columns (e.g. micro centrifuge dialysis devices)? As an alternative, I do agree with Gaurav also on trying to concentrate your protein as much as possible using a Vivaspin concentrator, and then resuspending in a different buffer if that´s what you need.
Good luck!!
What MWCO did you use? Odd-shaped proteins may be quirky during dialysis (i.e. they may leak through membranes that would contain globular proteins of the same mass).
Also, what protein conc. assay are you using? Perhaps there is an inhibitor in the dialysis buffer. Try checking pre-post dialysis aliquots in SDS-PAGE.
If you're unlucky, you might have a protein which binds to the membrane, so you will lose lots that way. I presume you aren't working on a cellulose binding protein?
Did you see precipitation? If not, you may have overestimated your protein concentration, perhaps due to imidazole? How did you determine the protein concentration - did you run a SDS gel to confirm purity and concentration?
Two important questions, what cut-off did you use and was your enzyme pure, partly purified or crude extract?
1. Has your dialyzed protein volume increased?
if it increased you diluted it, if it did not change then
2. Is there precipitation?
yes- that's where the protein is, no-protein is lost from dialysis tube (pore size, lose closure)
3. Was protein concentration overestimated? certainly possible with imidazole present
measure imidazole alone using the method you used for your starting protein concentration and subtract it from your earlier total. Imidazole has to be at the came concentration you used with your protein to be dialyzed.
As an alternative to the good suggestions already given, I find the Slide-a-Lyzer cassettes sold by Pierce, now Thermo Scientific, quite useful since it does not require tying dialysis tubing. They come in different mol wt cutoffs and different sizes. http://www.piercenet.com/cat/slide-a-lyzer-dialysis-cassettes
You could use a SEC chromatography resin to perform the buffer exchange but then the protein would need concentrated again. Acetic acid preciptiation or TCA of the same might work to concentrate or use a lyophilizer.
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