Odyssey system from Li-Cor for quantitative Western blotting
To quantify protein intensity from Western blotting, we found the Li-Cor Odyssey system shows superior capacity compared to regular ECL luminascence method as mentioned in the section \'Quantitative Western Blotting\'. The Odyssey scanner is available from Li-Cor Biosciences with a price around 50K dollars. Several points worth-noting when using the Odyssey scanner.
1. Although nitrocellulose membrane of any brand could be used, we find the one from Li-Cor shows low background and high consistence. Name: Odyssey Nitrocellulose Membrane, P/N: 926-31092.
2. When blocking, the TBS buffer without Tween 20 should be used.
3. When washing, the TBS buffer with 0.5% Tween 20 should be used. We use also use this buffer to make 3% BSA solution as primary and secondary antibody dilution buffer.
4. You may always re-use primary and secondary antibodies and store at 4C with 0.01% Azide.
5. When scanning, you may choose 84 or 42 as sensitivity to get a big image.
6. We have tested various secondary antibodies. And here is the best combination: IRDye 800CW Donkey anti-Rabbit IgG from Li-Cor, Cat-926-32213; Alexa Fluor 680 Goat anti-Mouse IgG, Cat-A21058 from Invitrogen.
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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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