How To Preserve Your Samples In Western Blotting
When running a quantitative Western blot, it’s crucial that your sample preparation is consistent. Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly. And after the protein extraction, it’s important to handle the samples in an identical manner to keep protein degradation from affecting them unequally. Here are some tips on how to handle and preserve your protein samples for Western blotting:
Work quickly
Working quickly can minimize potential damage to your samples by simply allowing less time for them to become degraded. If you are collecting samples that will not be processed immediately, flash-freeze them in liquid nitrogen as soon as possible — but make sure you are aware of liquid nitrogen dangers and ensure you handle it safetly in the lab.
Alternatively, process the samples immediately, and use or freeze the samples as soon as possible. It’s important to store processed samples at -80°C, since some protein degradation can still occur at -20°C.
Keep everything cool
Heat is the enemy of proteins in solution, since proteases are active at warmer temperatures. For this reason, keeping materials and samples cool will go a long way to preventing degradation. If you are grinding your samples with a mortar and pestle to extract proteins, pre-chill the mortar and pestle with liquid nitrogen to make sure they’ll stay cold. Always keep your tubes on ice, and consider working in the cold room to keep all your racks and other tools cool. Gels can be run in the cold room, too, and if you have particularly sensitive samples, you can pre-chill the running buffer in the running apparatus overnight.
Use protease inhibitors
Even being as cautious as possible, proteases may still affect your samples. As an added precaution, it is common to add protease inhibitors to your samples. Protease inhibitors like PefaBloc (aka AESBF) are sold individually, so you can target a class of proteases. A simpler approach is to use protease inhibitor cocktail tablets, which are available from many companies (like these from Sigma Aldrich), and inhibit a wide range of proteases.
Source: A great video by Agrisera that’s packed with tips for superior Western blotting.
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All the above are valid points if you are doing native western blots, but if you are running standard SDS-PAGE with denaturation and reduction of samples before loading, the best way to preserve protein integrity and to harvest total cellular protein is to lyse your tissue directly in 1X SDS-PAGE sample buffer, followed directly by heating at 70°C for 10 minutes. This irreversibly inactivates proteases, and results in complete destruction of all cellular membranes. After brief sonication to disrupt the DNA, you are left with a solution containing all cellular proteins as linear polypeptides which is stable at room temperature and can resist multiple freeze-thaw cycles.
The one disadvantage is that due to the presence of reducing agents in the sample (beta-mercaptoethanol or DTT), you must use a reducing-agent compatible assay for protein quantification. I prefer to use the Bio-Rad RC-DC kit (based ont he Lowry method) because it can tolerate relatively high concentrations of reducing agent. BCA-based assays (such as the Pierce BCA reducing agent compatible kit) are far too sensitive to reducing agent contamination to give reliable results.
A nice sample protocol for direct lysis of tissue is in this protocol published by Christer Ericsson and Monica Nistér (link is to a PDF):
https://www.biobanks.se/documents/Protein%20Extraction%2020030807.pdf
I would really urge everyone doing denaturing SDS-PAGE to use this protocol, it is by far the best way to minimize protein degradation and to extract total cellular protein.
One more thing, this also completely obviates the need to worry about using expensive protease inhibitors or a range of different buffers (e.g. RIPA, NP-40, etc.) All cellular proteins are efficiently denatured, and even membrane-bound ones or extremely hydropho0bic ones end up in solution.