Who know anything about dialysis on protein extractions ? - (Mar/15/2009 )

Hello

I think to do dialysis on protein extractions from brain tissues to get rid of the salts that currently cramping my westerns.
But i don't know much about how to do the dialysis, can you give me some tips on that, what do i need. Nobody in my lad use that.

Thanks,

Snoopy

Snoopy on Mar 15 2009, 03:51 PM said:

Pierce markets a variety of dialysis cassettes and tubes. Browse http://www.piercenet.com/products/browse.cfm?fldID=040101 for their products (or check your other favorite companies for similar products). These products are easy to use and come with literature describing how to perform dialysis. In essence, you will put your sample (e.g. nuclear extract) into the dialyzer and incubate at 4°C overnight in a stirring solution mimicking your extraction buffer but without the salt. Sometimes a second or third shorter round of dialysis is performed with fresh buffer to further reduce salt conditions, depending on the desired final concentration. The salts will then diffuse out through the dialyzer pores while the larger proteins, etc. stay behind in your sample. What you need to know beforehand is your sample size and desired protein size (remember small proteins often also diffuse out of the pores). Remember, if you did a protein assay to determine the concentration of your sample extract before dialysis, you will need to do another afterwards.

An other option is the use of Vivaspin ultrafiltration spin colums, useable for concentration and/or purification of biological samples. It's faster than dialysis just a few hours, depends on the concentration of the protein.

here are a couple of pdfs from pierce to explain dialysis:

what if my protein is 18KD is it possible not to lose him in the process ? Are there pores small enought to keep him and loose only salts?

Snoopy on Mar 17 2009, 11:42 AM said:

You will need to buy the right cassettes with the proper cut off for your needs. A variety of dialysis cassettes are available with cutoffs below 5 kD. Don't buy one with a 20 kD cutoff.

Thank u all guys a lot

Snoopy on Mar 17 2009, 07:42 AM said:

Hi

You will get a range of cassettes/membranes from 3kd to 1000kd , always try to use a membrane 3 times less size of your protein , i mean if ur protein is 18kd try to use 3kd or 5kd membrane to get better hold of your protein. (Membrane Manufacturer will tell size of the membrane based on globular proteins).

All the best
Regards
Sudhakar