Mona, along with protease inhibitor also supplement your lysate with phosphatase inhibitor cocktail.  Try to use it fresh and if have to store for later work aliquote the lysate.  

Low tempaerature such as 0-4 degree.

Fresh as soon as possible.

Buffer added with glycerol.

Although there might be other reasons, few aspects that can be taken care of and which I feel might be required for standardization

1) The temperature at which the buffer is prepared to adjust pH. Considering that RIPA has Tris Buffer (which is very sensitive to temperature fluctuation), you may have to adjust the pH at the temperature close enough to the point where you would like to store the protein lysate.

2) Change the buffer ( eg: to HEPES or CAPSO -it will be trial and error). Since you are storing a protein lysate and constitutes 'n' number of proteins along with your protein of interest, the degradation may be triggered in any of those proteins.

3) Increase the salt concentration in the buffer.

4) Raise the levels of glycerol used for lyophilization (~50%).

5) Try adding glucose to the buffer as it might help in stabilizing the lysosomal membranes (thus, avoiding protease release).

Hope so this is of some help in solving your problem.

Dear Monalee, 

To me, it does sound very surprising that you have such significant protease activity at -80 degrees, especially considering that you have added protease inhibitors and use RIPA buffer. I assume that you would like to use the lysate for IP's or downstream experiments where you need to keep your proteins in their native state - otherwise, you could of course store the lysates in a fully denaturing buffer. 

How do you thaw out, and treat, your samples after storing them at -80? I find it much more likely that, perhaps, this is the point where your degradation takes place. 

Best wishes & good luck, 

Roland.

Thanks Rohit .....been there and done those. No luck :(

Thanks Shixue...havent tried the glycerol.

Thanks Harsha ...will try those.

Thanks Roland...I thaw the aliquoted samples on ice. I have also stored the lysate in Lammeli buffer after heat denaturation and they still degrade. 

Dear Monalee, 

If you have heat denatured your samples in Laemmli buffer, I would hazard to say that it almost certainly is not proteolytic activity originating from a protease. I don't think there are any known proteases that will still be active after being boiled in SDS, and subsequently also stored at -80 degrees.

When you say that the proteins in the samples degrade, how do you see this? Do you look at Coomassie or Ponceau stain? 

/Roland.

Hi Roland,

When we carry out immunoblotting with fresh samples, the b-actin (which we use as loading control) appears as a single band at around 43kDa. We have also tried PTEN and TGFb1 and we see bands of appropriate MW. If we run the same samples a week later, the original bands get fainter and additional bands appear at around 10-15 kDa. If we re-run the samples 2 weeks later, the original bands totally disappear and we see much stronger bands at 10-12kDa.

We have not looked at these samples using Coomassie or Ponceau.

This has been happening repeatedly but only with GI samples, not muscle tissue or pancreas or stomach...just SI and colon.

Thanks for following up :)

Monalee.

Hi again Monalee, 

That is really mind-bogglingly weird - I guess, like you said, there must then be some sort of extremely stable protease that is present in the GI tract only. It's so odd though, I would have really thought that boiling the samples surely would inactivate any and all proteases, especially if done in SDS sample buffer. It would be really interesting to hear if you do figure out what it was, or how to get around it,  in the end - sorry for not being of more help!!

Best of luck, 

Roland.

Hey Roland,

Will let you know if we sort it out.

Thanks for taking the time to read the post and share your thoughts :)

Good luck to you as well.

Monalee