Laemmli Sample Buffer (5X)
- DTT powder (5g) from Fisher #BP172-5.
- To make protein loading buffer of 5X with reducing agent DTT (100 mM).
- Aliquot to 1 ml each and store at -20C.
1. Weight 6.45 g SDS, add 16.125 ml 1M Tris, pH 7, add H2O to ~30 ml in a 50 ml Falcon tube.
2. Swirl the tube very slowly. You may warm it to 37C if necessary. It will take a while for SDS to be completely dissolved.
3. Add the solution into the DTT bottle and dissolve all DTT out. Add H2O to 32.25 ml. Swirl slowly to dissolve the DTT.
4. Transfer all solution to a 100 ml beaker and add 32.25 ml 100% glycerol.
5. Stir slowly until homogenous. Transfer several time between the beaker and a 50 ml Falcon tube to help.
6. Adjust pH to 6.8. The addition of DTT will affect pH of the solution.
7. Add 0.0645 g bromphenol blue to the beaker.
8. Aliquot and store at -20C with 1 ml each.
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SIRT6 links glucose and lipid metabolism
SIRT1, a histone deacetylase, has drawn considerable attention due to its important roles in metabolic regulation and longevity. SIRT1 is induced by fasting and suppressed by feeding. SIRT1 increases glucose production and beta oxidation in liver to meet energy needs during food deprivation. Another member of SIRT family, SIRT6 follows similar expression patterns as SIRT1. The function of SIRT6 in metabolic regulation remains unknown.
Kim and colleagues found SIRT1 can regulate SIRT6 expression in liver. Specifically, SIRT1 functions together with FOXO3a and stimulates SIRT6 transcription during fasting. The activation of SIRT6 directly suppresses gene expression of metabolic enzymes involved in triglyceride synthesis and glycolysis in liver. Deficiency of SIRT6 causes increases of glucose utilization, reduced beta oxidation and as a result, fatty liver. More importantly, Kim et al found the expression of SIRT6 is decreased in human fatty liver samples which indicates SIRT6 may play critical roles in liver steatosis in clinical settings. This study landed in Cell Metabolism of this month.
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