Laemmli Sample Buffer (5X)
Reagents:
- 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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Autophagy hits heart
Autophagy is acutely upregulated to provide necessary nutrients during starvation for survival. Under normal condition, constitutive autophagy is critical to perform house-keeping functions to eliminate damaged organelles and dysfunctional long-lived proteins.
Ageing is associated with accumulation of dysfunctional proteins in cells. Autophagic activity is decreasing along with ageing. Studies in worms and flies show that reconstitution of autophagy can increase life span. A recent report by Taneike and colleagues explored the role of autophagy in age-related cardiomyopathy.
In heart, autophagic activity is decreasing when ageing. Taneike et al eliminated autophagy in heart by knocking out an essential gene, ATG5, specifically in heart. The researchers found deteriorated cardiac function at 10 months of age in the deficient animals compared to wild type controls. The dysfunction of autophagy causes accumulation of dysfunctional mitochondrial, reduced mitochondrial efficiency and significant oxidative damage in cardiomyocytes. More interestingly, evidence of mitochondrial damage is obvious as early as 3 months old, before cardiac remodeling and dysfunction manifest. This study points to a possibility that autophagy is critical in maintaining cardiomyocyte homeostasis and autophagy may be a target for future therapeutic design for heart failure....
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