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.


Comments

Add a comment
Posted by ebiomethods Mon, 10 Dec 2007 13:05:32
Subject: Comments are welcome

Site News
New feature:
You can add comments on protocols now. Just go to any individual method page and click on the "Add a comment" link.

Highlight of the month
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. ... Read more highlights.