precipitation of guanidine HCl-sample after adding 4X laemmli buffer - SDS-PAGE protein sample preparation (Apr/25/2008 )
I encountered a problem precipitation of protein occur after adding 4X Laemmli buffer.
The procedure:
1) I used 8M guanidine HCl to elute proteins from the streptavidin resin.
2) Elution of protein collected.
3) Add 4X Laemmli buffer before I load the SDS-PAGE
4) Protein precipitation occured...... cannot run SDS-PAGE.......
I tried with 5% skim milk with guanidine HCl and add 4x leammli also form precipitation.
Any idea how to neutralize the guanidine HCL in my sample before i add the Laemmli buffer?
or is there any way that I could add the 4X leammli buffer without forming precipitation?
The procedure:
1) I used 8M guanidine HCl to elute proteins from the streptavidin resin.
2) Elution of protein collected.
3) Add 4X Laemmli buffer before I load the SDS-PAGE
4) Protein precipitation occured...... cannot run SDS-PAGE.......
I tried with 5% skim milk with guanidine HCl and add 4x leammli also form precipitation.
Any idea how to neutralize the guanidine HCL in my sample before i add the Laemmli buffer?
or is there any way that I could add the 4X leammli buffer without forming precipitation?
Hi!
Just dialyse your probe against 6M or 8M urea in microdialyser ( or in cap of eppendorf tube) near 30-60 min . And mix with sample buffer. ( 6M Gua-Hcl precipitate in SDS -PAGE but urea solution not)
Hi, thank you for replying my post regarding the gua-hcl protein sample.
Can you teach me what is the reason for the precipitation? It is because the salt contain in the gua-HCl which lead to salting out of the protein sample?
Thank for replying again.
Regards,
Leyhian