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Protein agregation in WB samples


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#1 jopa

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Posted 29 July 2011 - 01:33 PM

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

#2 bob1

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Posted 30 July 2011 - 08:38 PM

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.

#3 jopa

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Posted 31 July 2011 - 01:56 PM

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

#4 bob1

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Posted 31 July 2011 - 04:00 PM

In the 1x form.




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