Disadvantage of crossflow filtration - (Jun/16/2015 )

Dear,

I'm working on purifying recombinant protein. I passed filtration step by crossflow filtration to concentration my protein. After electrophoresis, I see that the sample after filtration was crumbled and it seem so dirty compared to the sample before filtration. This problem caused my target protein binding to the column not good. Many protein attend in flow through applied.

I wonder that the crossflow filtration process effect on my protein, so it can't binding onto the column?

Some information:

Target protein: 34kDa.

Crossflow filtration column: 10kDa.

Rate of pump: 250 rpm.

Time of process: 3 hours.

have you determined (eg by immunostaining) that the protein in the area of 34 kDa is indeed your protein of interest? there are a lot of proteins around that size.

what i see in your stained gel is that the "after" is more concentrated than the "before" but contains the same components, not "dirtier", and, if the band showing in all three lanes is your target protein, that it didn't bind to your second (binding) column. however, when using a binding matrix specific for your protein, you should expect the flow through to contain a lot of components (everything but the protein of interest). keep in mind that there is also a limit to the amount of protein that the column can bind and may have been overloaded.

have you tried eluting your protein of interest from the second column? if so, then how does it look on the gel?

also, by "crumbling" i assume you mean that you saw a precipitate after crossflow filtration. this could be caused by changing buffer conditions, in which case you might be able to redissolve the protein in the original buffer, or by physical harm to the proteins, in which case you should be able to solubilize with sds-page sample buffer and look at with the gel.

"Crumbled". I mean my proteins were broken to many pieces, so the stained seem "dirtier" than "before" sample.

After this problem, I tried applied sample on column without filtration. Unexpectedly, all the target protein binded on column. Therefore, I don't understand why the filted sample binding bad than the sample which without filtration.

maybe the protein was denatured by the process.

you say that the proteins were broken into many pieces. that sounds like you haven't inhibited proteases and, in the time it takes to filter, the proteins are being degraded.