Rafael Garcia wrote:
> I am solubilizing difficult proteins using a pretty harsh extraction solution:
> 7M Urea
> 2M Thiourea
> 0.05 M DTT
> 0.01M Tris
> 2.5% SDS
> 1% N-lauroylsarcosine (detergent)
> protease inbitor cocktail
>> Something in this mixture makes SDS-PAGE gels look streaky and dark.
Urea and thiourea can cause problems in SDS-PAGE, especially at high
concentrations. Also the presence of high concentrations of non-ionic
detergent can interfere with SDS-binding to the protein.
In addition, it might be better to solubilise at the pH of SDS-sample
buffer (6.8) instead of 7.4. You may be interfereing with the stacking
effect of DISK-electrophoresis (see the original Ornstein-paper from
1962 for a detailed explanation). This too would result in broad bands.
Are you boiling your sample before electrophoresis? This can interfere
with solubility, try 10 min at 60 degrees instead or even, since you are
working with urea, 30 min at 37 degrees.