I would like to use relatively old lysate in an experiment (10 years old lysate).
Because some of my antibodies are reconizing full length proteins, but also fragments and oligomers, I want to evaluate the general quality of my samples looking at the protein quality. That would allow me to be sure that my group is homogeneous in term of storage condition.
Hi there,
As you have antibodies, you can check the behavior of the protein on native and/or SDS/PAGE and WB... And compare with how the protein behaved prior storage.
If your protein is an enzyme, and it has an activity that is specific enough to measure in a crude lysate, you could measure the specific activity of the enzyme.
Hi Nicolas, the better way is to measure activity if you have some idea that the rest of the proteins do not interfere with the measurement...beyond that, 10 years is a long time to expect a good enough quality of your protein, e.g. proteases, oxidation, deamidation, etc., i would use it carefully
Hi Nicolas,
If you want to carry out a WB, the first thing I would do is just to check the degradation on a membrane after electrophoresis with red ponceau or in the gel with coumassie blue.
Thank you for all these ideas!
Comparison of running behavior in western blot is not so simple. First observation is that some proteins ran as usual (like microtubules) and some other ran a bit funny (some soluble proteins like enzymes). Therefore it is not simple to evaluate (and even quantify) how much lysates supported the long storage to confirm the homogeneity of the group.
Drastic change in protein activity may be indeed a solution to remove the worst samples. Thank you for the suggestion, I will give it a try.
I also agree with you Diego; however these are unique samples so if I could still analyze them knowing that no obvious artifacts come from these samples it would be fantastic.
Finally, tissue was stored in -80°C, so I cannot compare results from previous western to compare results. I will take extra care of the protein distribution on the membrane, but as far as I remember, after transfer membrane was looking "as usual".
Hi, If the protein is enzyme you can try enzymatic activity by specific substrates (I suggest fluorescent one) and calculate specific activity. Otherwise you can try Lowry or Bradford assay depending on solubility or membrane-bound types.
Cheers, A.
As i understand you want to know the quality of the protein in stored sample. Exact mechanism to know the quality of protein i did not know,, but i have the suggestion to check the quality (protein is available and equally available) you can do the estimation assay for protein. Firstly check your sample for the concentration of the protein using bradford assay protein estimation. this will give your protein estimation in different sample after so long storage. once you have estimated your protein then you can do WB for protein and the result will be depend on your antibody
You can check the quality of protein in lysate by SDS-PAGE, 2D electrophoresis, western blot etc. Protein could be degraded due to the presence of proteases in the lysate. Also, microbial contamination might degrade proteins.
If the lysate was stored in -80C through out that 10 years, I think it shouldn't be a prob. But if there was thawing along the way, degradation would have happened. You can check on it through SDS-PAGE. However, I doubt it would be plausible for functional analysis of proteins in that lysate.
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