Some protocol adds reducing agent to Laemmli sample buffer and store it at -70 or -20 degree Celsius. Some adds it fresh before boiling with protein sample.
Have you guys ever tried both add reducing agent fresh and not fresh? Does it result differently in Western blot or gel electrophoresis?
Thank you
The classical protocols indeed say to add fresh BME and DTT. A typical example is the Bio-Rad manuals available. I once worked in a lab that routinely used a Laemmli Buffer ready stored at -20°C. I used this buffer for a long time and never had a problem with my gels. However, I never compared both methods to check if, in my samples, this could make a big difference or interfere in any way.
It would depend on your downstream application. Both decompose with time, more so when diluted and faster in some buffers than others. Decomposition products of DTT can form adducts with proteins that may complicate mass spec. Beta-mercaptoethanol can cause artifacts with SDS-PAGE in the 55 kDa range when using very sensitive detection methods like silver staining but this shouldn't be a problem for Western blotting. For basic SDS-PAGE and Western blotting, you can add Beta mercaptoethanol to your sample buffer several weeks in advance without a problem if you store it at 4o C (I discard mine after about a month). If you choose DTT, I would discard it after a week.
We freeze our Laemmli buffer with BME at -20°C all the time to store it for >6 months and had never any problems with it.
My lab stores ~ 10 1mL aliquots of 4x Sample Buffer with BME [20mM final] at -20C for several months, too. I searched for this answer because my colleague and I both notice that--after several uses (freeze/thaw) of the same aliquot AND first uses of different, unused aliquots--we can no longer smell the BME in the 4x Sample Buffer.
I first noticed this when I was nearly out of an aliquot of 4x Sample Buffer...so I thought maybe the volume was too low to have a significant odor. But now we're noticing the same thing with full 1mL aliquots after 1-2 months at -20C.
Obviously, the best suggestion for me would be to make the BME fresh each time. But I wanted to know if anyone had any insight as to why this is happening.
Thank you!
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