Smearing of protein samples from whole tissue lysates - (Mar/06/2006 )

I am lysing mouse tissue samples using a dounce homogeniser in 0.5ml lysis buffer : (10%SDS, 2% Triton X100, 5M Urea, 100mM DTT and 5mM EDTA). Samples are sonicated and Gel Loading Buffer added (10% glycerol 50mM Tris pH6.8 0.2% Bromophenol blue. Samples are boiled and run on 8% SDS PAGE.

Samples resolve poorly and I get smearing (probably from DNA).

Any suggestions on how to overcome the smearing?

-peteri-

you should add protease inhibitors in your lysis buffer.

-fred_33-

QUOTE (fred_33 @ Mar 6 2006, 06:09 PM)

you should add protease inhibitors in your lysis buffer.

Are you sure that you need protease inhibitors if you lyse in denaturing buffer? Just a question.

I solubilize E coli pellets in laemmli buffer without any protease inhibitor. i have nice bands, proteases cannot work if they are denatured.

I also had some troubleswith these samples, I was loading a tris-tricine gel, and it was smearing. Finally I got better results on tris-glycine !
I was also thinking that it was the DNA?
May be you could add some DNase (1ug/mL) 30 minutes on ice. However I never worked on tissue dounce homogenized.
If someone else has a better idea...

-Missele-

QUOTE (fred_33 @ Mar 6 2006, 07:09 PM)

you should add protease inhibitors in your lysis buffer.

Given the high Urea content and DTT, I thought proteases would be denatured / inactivated???

-peteri-

Yes I would say also that proteases cannot work in denatured buffer.
neither DNase, that's why you should forget my previous post. Stupid mistake.

-Missele-

QUOTE (Missele @ Mar 7 2006, 12:09 PM)

Yes I would say also that proteases cannot work in denatured buffer.
neither DNase, that's why you should forget my previous post. Stupid mistake.

Boiling samples at too high temperature too long, or loading too much of the sample on the gel can cause smearing.

-mickihiiri-

I have same suggestion as miselle. You might have better luck with a different resolving gel system. Also as another person suggested, boiling time could also affect the protein banding.

-daisy_d-