doublets on 2D gel - (Mar/09/2016 )

Hi,

What would cause the "doublets" that I'm seeing on my 2D gels?

thanks

-biochem06-

i would think that would be proteins with similar pI and a little different molecular weight.

you can check them on mass spec.

-mdfenko-

I remember reading somewhere (but can't find it now) that it can be an artifact. Due to placement of the strip on the gel?

-biochem06-

Ah, I just found something, although it's still unclear why it happens.

From - http://www.bio-rad.com/LifeScience/pdf/Bulletin_2651.pdf

3. Twin vertical spots or vertical doublets throughout the gel.

This effect is sometimes seen when thiourea is included in the sample solution. Its cause is unclear.

-biochem06-

My GUESS (from very limited background of OChem) is that the sulfur in the thiourea reacts with a population of the protein, on the cysteine, to form a "post-translational modification" disulfide bond that modifies the molecular weight of the protein.

What is the concentration of thiourea in your sample? Any chance of calculating the molar ratio of thiourea/protein to see if we're using all the thiourea up and have excess protein?

Admins feel free to tell me I'm crazy.

-Michael Starr-