Protein agregation in WB samples - (Jul/29/2011 )

I've been lysing my cells with RIPA to do wb and I generaly use this protocol

- tranfect cells with construct (>80% transfection)
- 48h after I lyse with RIPA + protease inhibitors (100ul per well in a 24 well plate)
- I spin at transfer supernadant to new tube
- Add 5X sample buffer +DDT in a 5x dilution, boil and load

Question is:

How much DDT should I have in my final ready to load sample?
If I want to save my samples should I do it before or after adding the sample buffer?

Sometimes i've noticed that if I use samples that have been at -80 for a couple of days, I do a 1 minute spin to remove agregates and I have a big pellet in the tube (sample in RIPA and sample buffer) and in the western i've lost my bands. I should get 2 bands since my protein is cleaved. Sometimes I get the band of the precursor but not the cleaved form and other times the other way around which is weird since if I have the cleaved form I should have the precursor.

I think I have protein aggregation, thats why I have the big pellet, but I don't know how to prevent it. I've tried sonication of samples and boiling before loading and still the same problem.

Any suggestions... sorry for the big text

The DTT should be about 150 mM (IIRC). You can freeze your samples before or after adding sample buffer, it shouldn't matter. You will need to re-boil your samples post thawing if you have stored them in sample buffer.

Okay so its 150mM in my 5X sample buffer solution or 150mM in my final sample with the sample buffer e a 1:5 ratio.

thanks

In the 1x form.