Western Sample Buffer - DTT vs B-mercaptoethanol - (Mar/08/2013 )
I have a few questions regarding DTT and B-mercaptoethanol used in sample buffer for western blot.
<*>If I wanted to prepare my protein sample in buffer with reducing agent, heat and load then freeze remaining to be used within the next week or so which reducing agent would be a better choice - DTT or B-mer?
<*>Do I need to re-heat these samples after thawing or just thaw, mix and load?
<*>In general, best temperatures to heat samples: DTT 70C and B-mer 90-95? Or both can be heated to 90-95C?
Either is fine, basically they both work out at the same molarity of reducing molecules despite being different concentrations (DTT has 2 S; B-ME, 1). DTT tends to go off quicker in storage (stock solution), but smells less (IMO) than B-ME.
In theory you don't need to re-heat the samples after freezing, but many people re-boil them.
Both are fine at 90-95 or 100. However, the temp you should use depends on the protein you are looking for, some are damaged by high heat.
How many times can you freeze thaw these samples in buffer? 3 or 4 times? I understand you begin to lose protein to degradation over many cycles of freeze thaw but I was curious what a safe number of times would be. It probably depends on the protein too.
It definitely depends on the protein, things like actin will last several (10?) but others won't even last 1 cycle.
At what temperature is the most ideal to store prepared protein samples? -20C? -80C? and for how long?
They will keep indefinitely at -80, for several months (protein dependent) at -20. Purified proteins can be kept at 4 for quite some time (again, protein dependent) if preservatives are added to the solution.