Quantitative Western
The common enhanced chemiluminescent (ECL) method of Western blotting has many disadvantages. We have been using fluorescent dye conjugated secondary antibodies for WB and use the scanner from Licor to quantify the signal. We found the method is very accurate, convenient and reliable. More importantly, you can use two different colors on the same membrane for two antigens. The following antibodies have been used successively for quantitative fluorescent WB.
Note: if you are not satisfied with the signal intensity, you can dry the membrane before scanning and enhance the signal by 3-4 fold without changing the background.
1. IRDye 800CW Goat Anti-Rabbit IgG, #926-32211 from Li-Cor. We use 1:5000 to 1;10000 dilution after reconstitution. This dye will light green bands in the scanner.
2. Alexa Fluor 680 Goat anti-Mouse IgG, #A21058 from Invitrogen. We use 1:5000 to 1;10000 dilution. This dye will light red bands in the scanner.
3. Alexa Fluor 680 Donkey anti-Goat IgG, #A21084 from Invitrogen. We use 1:5000 to 1;10000 dilution. This dye will light red bands in the scanner.
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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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