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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Metformin: New mechanism of old drug
Metformin, one of the most effective antidiabetic drugs, remains on market for a long time. Yet, the mechanisms of action are still elusive. Past studies point to LKB1/AMPK axis as the mediator of gluoneogenesis regulation. A recent report challenges this notion and provides evidence suggesting metformin can function in the absence of LKB1/AMPK.
Foretz and colleagues found metformin suppresses glucose production in wildtype primary hepatocytes dose-dependently. To their surprise, this suppression remains effective in AMPK deficient hepatocytes. They went further to show there is no difference between wildtype and AMPK liver specific knockout mice for glucose regulation by metformin. Similarly, LKB1, the upstream kinase of AMPK, is not required for glucose lowering effects of metformin in hepatocytes and in animals. More importantly, Foretz et al found metformin treatment reduces energy state of primary hepatocytes and in livers of mice, which is probably the primary reason of decreased glucose production.
The new mechanism proposed by Foretz et al raises number of interesting questions, such as the target of metformin, the mitochondria changes after treatment, beta oxidation, glycolysis. Answers for them will provide more insights about the action of metformin and clear understanding of type II diabetes.
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