Among possible reasons you might want to consider:

1)  Even short exposure of your protein during or after Ni-NTA at room t° some contaminating proteases might lead to proteolytic digestion of your protein, resulting in smaller MW peptides which might be lost in dialysis with 3 kDa cut-off. Try to use Protease Inhibitor Cocktails at all steps of the cell lysis and protein purification.

2) Depending on your dialysis buffer, it looks like your protein might be prone to aggregation after removal of urea resulting in formation of high MW aggregates which might even not getting into the gel. Try to run western blot with your samples before after dialysis, and look for any high MW immunopositive bands. If you find them, try to use urea to keep your protein as a monomer.

Among possible reasons you might want to consider:

1)  Even short exposure of your protein during or after Ni-NTA at room t° some contaminating proteases might lead to proteolytic digestion of your protein, resulting in smaller MW peptides which might be lost in dialysis with 3 kDa cut-off. Try to use Protease Inhibitor Cocktails at all steps of the cell lysis and protein purification.

2) Depending on your dialysis buffer, it looks like your protein might be prone to aggregation after removal of urea resulting in formation of high MW aggregates which might even not getting into the gel. Try to run western blot with your samples before after dialysis, and look for any high MW immunopositive bands. If you find them, try to use urea to keep your protein as a monomer.

The protein my have been degraded by proteases.

Yes either your protein is degraded during dialysis or aggregated. However in later case you should see a pellet. Use enzyme inhibitor like PMSF during rupturing of cells and take proper care not to handle with bare hands, use globes and do purification at 4C. If it still doesn't improve you can take a time course sample form dialysis bag and analyse by SDS-PAGE to check the degradation or aggregation!

#Viktor #Adam #Chibuike #Shubhendu Thank for serious concern in my research issue. Here I like to bring in a notice that, previously I used to use protease inhibitor cocktail but didn't solve the problem so again I shifted to PMSF and benzamidine. Also, during dialysis, I can't see so much of pellet (no aggregation). It's very minute pellet. Once during this experiment,  I got to see protein band after the dialysis only in the supernatant (SDS-PAGE: Single band and Nanodrop: 4mg/ml). The pellet was very minute. I started concentrating protein (10ml) using 3kDA centricon. Again, I missed all my proteins which were really good in amount. Further, I process all my experiment in 4oc. Even I feel my protein is getting degraded but am not able to troubleshoot on it.  I can't see any protein(high molecular weight protein aggregates) stuck in the stacking gel. I did western blot but couldn't detect protein in after dialysis sample but detected in before dialysis sample.

Thanks a lot.

Sometimes during concentration of protein with centricon or amicon units, the protein may stack on the membrane of the concentration unit. Once precipitated, you will not be able to see it or recover it. instead of concentration, you can use gel-filtration with a column. This may help you.

It may be that your protein is being stabilised by the urea in the elution buffer and that a step gradient dialysis to refold your protein may prove beneficial (and stop it crashing out of solution). I have previously done a step gradient from 8 M urea to 6 M, 4 M, 2 M, 1 M, 0.5 M, 0.25 M then to buffer of choice (mine was PBS), this prevented loss of my protein due to aggregation. Each dialysis step was for a minimum of 4 h at 4C (not quick but quicker than starting the prep again).To save on some urea I made the 6 M initially and then diluted this with PBS to make the new concentration.

If on the other hand you are sure that your protein is soluble in the absence of urea and imidazole I would recommend you try using size exclusion chromatography instead of the dialysis step. This will act as a polishing step in your purification and also act as a buffer exchange.

Good luck

Hey, Please do a favor, and keep an aliquot of protein. Doing so, you can compare the effect of dialysis. Similarly, do not dialyze all the protein in one go. You can do short experiments, such as step dialysis as suggested by Rebecca.

Similarly, use the dialysis tubings from another supplier to see the effect. Some dialysis tubings require incubation of tubings in the boiling water to remove the preservatives. Please read about the tubings, and follow the suppliers instructions. You can also use dialysis buttons or Slide A Lyzer from Thermo scientific.

What i understand, something is happening during the dialysis. Therefore, i shall not go with further experiments after this dialysis step, including the concentration of the protein after dialysis. This shall add one more problem to understand.

Please do let us know about the outcome.

Best wishes!

Incomplete Dialysis can be an issue as well. Try few time points for dialysis and adding protease inhibitor (for example 0.5mM PMSF in Isopropanol) in the dialysis buffer can help.

Good Luck!