19 replies to this topic

[rpjkmust916]

Inmost sun, on 19 February 2011 - 07:03 AM, said:

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

Thanks Inmost sun for your suggestion...unfortunately in my lab only Comassie blue stain available....

May I ask about the electroelution?
I've read several papers about electroelution...looks like it require some specific apparatus. Is there any simple method that I can use to run electroelution?
Any suggestions, please?

[mdfenko]

rpjkmust916, on 22 February 2011 - 04:24 AM, said:

Inmost sun, on 19 February 2011 - 07:03 AM, said:

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

Thanks Inmost sun for your suggestion...unfortunately in my lab only Comassie blue stain available....

while silver stain is very sensitive and qualitative, it is too variable between different proteins to use for quantitation (and, therefore, percent purity). coomassie brilliant blue r-250 (not as variable as g-250) is well suited for this purpose. you may need to load more to see minor contaminants but they are minor and will not significantly influence percent purity determinations

Quote

May I ask about the electroelution?
I've read several papers about electroelution...looks like it require some specific apparatus. Is there any simple method that I can use to run electroelution?
Any suggestions, please?

all you need is dialysis tubing, horizontal gel apparatus and a suitable buffer

[rpjkmust916]

Thanks mdfenko,

Can I use the running buffer that I've used to purify the protein with IMAC column to do the electroelution?
Or do I have to find a different type of buffer?

May I know, how much the concentration of target protein that you've usually recover from electroelution?

Thank you very much for spending time in answering my questions.

[mdfenko]

rpjkmust916, on 22 February 2011 - 05:34 PM, said:

Can I use the running buffer that I've used to purify the protein with IMAC column to do the electroelution?
Or do I have to find a different type of buffer?

standard electrophoresis buffers work well, others you may have to try yourself

Quote

May I know, how much the concentration of target protein that you've usually recover from electroelution?

concentration depends on how much protein is in the band being eluted and the volume of buffer in the dialysis bag. you can recover most of the protein from the gel.

[rpjkmust916]

Thanks mdfenko  

Edited by rpjkmust916, 23 February 2011 - 09:08 PM.