4 replies to this topic

[dnafactory]

Dear all,
I'm using the Microcon concentration devices but I recover only a small amount of the starting maerial.
I loaded the sample dissolved in 8M Urea (the concentration was at least 1ug/ul) into the YM-30 device; I centifuged at 14000g for 6min and only 15ul were left. I put the column upside down and spun at 1000g for 3min. I could recover only 6% of the total protein amount.
How can I improve the recovery? Can anybody help me, please?
Thanks in advance

[mdfenko]

what is the mw of your protein? if it is near the cutoff of the microcon then you may want to use a lower cutoff microcon.

also, since the protein is in urea, it is denatured and won't act like a normal globular protein (assuming that the protein is normally globular) and may pass through the membrane anyway.

[dnafactory]

mdfenko, on Jun 19 2009, 05:08 PM, said:

what is the mw of your protein? if it is near the cutoff of the microcon then you may want to use a lower cutoff microcon.

also, since the protein is in urea, it is denatured and won't act like a normal globular protein (assuming that the protein is normally globular) and may pass through the membrane anyway.

Hi mdfenko,
first of all, thank you so much for your reply    I'm trying to concentrate whole cell extract, not a specific protein. Your observation about the 8M Urea is really good and I hadn't thought about the fact that unfolded proteins may not be retained. I'll try by using another buffer.

I decided to lyse in Urea because I need to run 2D gels with those proteins and I cannot have them resuspended with detergents or Tris. Do you have any suggestions about a buffer I could use?

Thank you again!

[dnafactory]

Despite the fact they write 8M Urea is compatible with Microcon, I could manage to concentrate BSA in PBS but not in Urea... With Urea, BSA went to the flow-through  

[mdfenko]

dnafactory, on Jun 22 2009, 04:49 AM, said:

I decided to lyse in Urea because I need to run 2D gels with those proteins and I cannot have them resuspended with detergents or Tris. Do you have any suggestions about a buffer I could use?

why can't you suspend the sample in tris or detergent? tris in the sample should have little to no effect on ief.

nonionic detergents can be added to the ief gel so that the protein doesn't precipitate when separated from the sample buffer during the run. just wash out the nonionic detergent before (or during) the sds incubation prior to the second dimension.

Quote

Despite the fact they write 8M Urea is compatible with Microcon, I  could manage to concentrate BSA in PBS but not in Urea... With Urea,  BSA went to the flow-through

the urea won't have any (or significant) effect on the membrane. the protein is another story.