High MW smear - how can I get all protein into the gel? - (Nov/08/2007 )

Hey Everyone!

I have a problem when I run Western blots. My protein of interest has a MW of ~180kDa and is located in the plama membrane. I am always able to detect my protein, however in 90% of my blots the antibody also detects a big smear on top of the gel, which easily runs higher than 400 or even 500 kDa (just a guess, since the marker does not go that high).
I noticed that this smear appears if I heat my sample. In order to avoid it (and to get all sample into the gel) I now try to keep the sample cold at all times, which seems to work better. But sometimes I still get the smear, especially when I isolate protein from a lot of samples. So, apperently the samples (if too many) get too warm at a certain time.

I know that my isolation procedure is crucial, but I am wondering if anyone here knows how to dissolve the aggregated proteins in the samples from which I already know that my protein will be stuck on top of the gel (heating apperently does not work... )

The samples are in RIPA buffer and just before loading I add cold 4x Laemmli buffer.

I attached two gels. The blue one shows two samples with a smear and three without (protein isolation on different days). The gray film shows how intense the smears sometimes gets.

Thanks a lot!

Hazel

for how long do you heat your samples?

if you heat too long you can cause your protein to coagulate. you can check by spinning your sample after heating.

also, you may be overloading your sample. you can try adding a little extra sds (and maybe a little more 2-me or dtt).

I don't heat my sample at all anymore. I load them basically straight from -80°C onto the gel.
As far as overloading is concered: I don't think the smear is a problem resulting from the amount of protein, since I almost always see that smear, regardless if I load 30µg or 150µg...

How much extra SDS, 2-ME or DTT would you recommend?

Also, has anyone any experience with bond-breakers in that respect?

Thanks,
Hazel