Hello, I hope analytical chemistry people or biochemistry major fellows could help me. Kindly advise on how to prepare the following denaturation buffer: [2% SDS, 1 M β-mercaptoethanol (β-ME)]?
The buffer is expected to be used with Endoglycosidase H enzyme, extracted from Streptomyces plicatus, CAS 52769-51-4 | 324717.
2-mercaptoethanol (or β-mercaptoethanol) is a liquid with a concentration of 14.2 M. It is very smelly, so use it in a fume hood, if possible.
Sodium dodecyl sulfate is a solid. The dust from SDS can irritant the lungs, so it's a good idea to wear a filter mask when weighing it.
2% in this context means 2 grams/100 ml of solution.
For 100 ml of the solution, you need 2 grams of SDS and 7 ml of β-mercaptoethanol (1 M/14.2 M x 100 ml). To somewhat less than 90 ml of distilled water in a beaker, stirred with a magnetic stirrer, add the β-mercaptoethanol and dissolve the SDS. Bring the volume to 100 ml with distilled water.
Make fresh or keep as frozen single use aliquots as β-mercaptoethanol degrades at room temperature.
If you want to prepare dentraration buffer with a total volume of 100 ml. First, add your solid soluble, so add 2 g of SDS to the empty bottle. Then add BME; usually pure stock of BME has a molarity of 14.3M, so to make it 1 M dilute it 14.3 times (use the formula c1v1=c2v2;e.g.: 14.3 Mx v1= 1 Mx 100 ml =6.99 ml)
add the 6.99 ml of BME to your 2 g SDS (in the fume hood), then, add solvent e.g: water to make up the total to 100 ml.
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