Hi guys, so in order to prepare stripping buffer to strip my Western blot membranes, I need to use DTT. We have in the lab DTT stored as 1 ml aliquots, and frozen at -20. I already thawed one aliquot three times, and refroze it again, since i only need 125 micro liters for the stripping buffer. My question is from your practical experience, how many times can I thaw and refreeze the DTT aliquot before it loses its activity. Many thanks.
The stability of DTT is more dependent on the time it is kept at room temperature (or low non freezing temperature) then on the actual number of freeze/thaw cycles. This is because DTT is not a large polymer (like proteins or nucleic acids) that can break due to physical shearing forces during thawing/freezing. The recommendation to make fresh DTT dilutions from frozen aliquots originates from the fact that when diluted to mM range it can't be kept for longer than a day at 4 degrees Celsius. I don't think freezing and thawing 3 times causes any problems if you do it quickly, but protein chemists are often very conservative with respect to their reagents and in my case I avoid freezing and thawing by making two types of aliquots, larger aliquots for long term storage, and small aliqiots for single use. So if your routine quantity is 125 microlitres, I would make 10mL of your DTT stock, from which I would make 10 aliquots of 130 microlitres, and keep the rest in large 1.5 mL aliquots. When the small aliquots run out, I would use a new 1.5 mL aliquot, use 125 microlitres for stripping, and aliquot the rest in 130 microlitre aliquots. This way you will maximally have two freeze and thaw cycles, without having to occupy large amount of freezer space for all the aliquots.
The stability of DTT is more dependent on the time it is kept at room temperature (or low non freezing temperature) then on the actual number of freeze/thaw cycles. This is because DTT is not a large polymer (like proteins or nucleic acids) that can break due to physical shearing forces during thawing/freezing. The recommendation to make fresh DTT dilutions from frozen aliquots originates from the fact that when diluted to mM range it can't be kept for longer than a day at 4 degrees Celsius. I don't think freezing and thawing 3 times causes any problems if you do it quickly, but protein chemists are often very conservative with respect to their reagents and in my case I avoid freezing and thawing by making two types of aliquots, larger aliquots for long term storage, and small aliqiots for single use. So if your routine quantity is 125 microlitres, I would make 10mL of your DTT stock, from which I would make 10 aliquots of 130 microlitres, and keep the rest in large 1.5 mL aliquots. When the small aliquots run out, I would use a new 1.5 mL aliquot, use 125 microlitres for stripping, and aliquot the rest in 130 microlitre aliquots. This way you will maximally have two freeze and thaw cycles, without having to occupy large amount of freezer space for all the aliquots.
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