Hi there!
A simple question to you; i have 10X DTT stock (500mM), and 4X laemmli stock. How much DTT should i add to my sample buffer? DTT should be diluted from 10X to 1X in the Laemmli buffer, or in the final SAMPLE?
So should i just add 100 ul of 10X DTT to 900 uL of 4X laemmli to have 1 ml of 4x final buffer (which will be then diluted with 3 parts of sample) ?
So for example, lets say i have 12 ul of sample, i add 4 ul of 4x laemli/DTT (in which i diluted my DTT to 1x), or should i add 2,8 ul of 4X laemmli plus 1,2 ul of DTT10X ?
Thank you everyone
Dear GianMarco
I suggest to you to dilite DTT in laemil, so you can prepare a small stock to be addedd to all the samples and you speed up the sample preparation.
Normally i'm using final DTT 25mM and it is enough to reduce well all the proteins.
However if you would like to use around 50mM fimal, you can prepare a 3X laemil stock by mixing mix 3 volumes of Laemil 4X with 1 volumes of 10X DTT in this way at the end you DTT will be diluted 9 times and it will be around 50mM.
best
Manuele
thank you, very nice, although in this way it is nearly 50mM in the final buffer, but then it gets diluted 2 more times when added to the sample, am i right?
Dear Gianmarco Matrullo
if you mix 3part of leamil 4X with 1 of DTT 0,5M you will obtain a 3X leamil-DTT those contain 500/4 --> 125mM of DTT.
This is a 3X stock that means that you have to mix 1 part of it with 2 part of sample (eg 5ul of leamil-DTT 3X with 10ul of sample) and you will obtain a sample with 125/3= 41,6mM of DTT.
Best
Manuele
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