Dear Community,
I recently performed a Western Blot on phospho-Erk1/2, phospho-STAT5, phospho-p70-S6K and phospho-STAT3. But the results were very strange. They all showed multiple extra bands and the bands on the correct height were not really making sense (in fact things I blotted before dozen of times with the same result now showed the complete opposite and strange superintense bands occured). I worked with all ingredients before for months without any problems (no extra bands, repeatable results, same protein amount). The only thing I changed was to store both prepared Mastermix (Lysate + Lysis Buffer for Dilution + 1/4 Laemmli 4x) and lysates at -80°C before blotting instead of -20°C. Can this explain my problems?
And if it is the reason can I prepare fresh mastermixes from the frozen lysates stored at -80°C or are they also damaged?
Thank you very much!
Yes, both protein lysates and Western blot master mixes can typically be frozen at -80°C for long-term storage without significant loss of quality, although some precautions should be taken.
For protein lysates, it's best to aliquot them into smaller volumes before freezing to avoid repeated freeze-thaw cycles, which can degrade protein quality. Additionally, make sure to include a protease inhibitor cocktail in your lysate buffer to prevent protein degradation during freezing and thawing. For Western blot master mixes, it's also a good idea to aliquot them into smaller volumes to avoid repeated freeze-thaw cycles. Ensure that all components of the master mix are compatible with freezing and thawing, as some enzymes or reagents may lose activity upon freezing. Labeling each aliquot with the composition and date can help you keep track of your stocks.
Overall, proper aliquoting and storage at -80°C can help maintain the integrity and functionality of both protein lysates and Western blot master mixes for extended periods.
Thank you very much, I already made aliquots and used Protease Inhibitor but it is very important to know that the storage conditions are not the reason for my problems!
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