3 replies to this topic

[Nay]

I am after a protein quantitation method which is ok to use when there is detergents in your solubilisation/lysis buffer. I would normally use the Biorad protein assay (Lowry assay), but I am getting massive errors (always increased estimates) of my protein concentrations. I have been using trizol to extract the protein so I assume it is due to a carry-over of phenol (when I don't use trizol, my lowry assay works well) - is there a quantitation assay which I can use if I have some phenol contamination?

I am sick of trial and error loading!

[ros]

Nay, on Mar 30 2005, 07:18 PM, said:

I am after a protein quantitation method which is ok to use when there is detergents in your solubilisation/lysis buffer.  I would normally use the Biorad protein assay (Lowry assay), but I am getting massive errors (always increased estimates) of my protein concentrations.  I have been using trizol to extract the protein so I assume it is due to a carry-over of phenol (when I don't use trizol, my lowry assay works well) - is there a quantitation assay which I can use if I have some phenol contamination?

I am sick of trial and error loading! 

<{POST_SNAPBACK}>

this might not help - but do you have to use Trizol? in my experience, Trizol is more trouble than it is worth for both RNA and protein! I would suggest changing your isolation protocol.

[Nay]

my antibody is horribly fussy and only seems to work when I use trizol to extract my proteins - I have decided to try dialysing my samples and/or using a buffer exchange column before I quantitate them now...

[amj2]

you can try this-


http://biology200.gsu.edu/core/Manuals/2D%...%20protocol.pdf


Hope it helps