Hi all!
I would like to perform a western blot to determine the amount of collagen type I and type III in human cardiac tissue biopsies. I am completely new in this technique so I am wondering if exist a specific protocol to get the best results.
Many thanks in advance!
Laura
The below protocol is from a recent publication (PMCID: PMC3925009). It should give you the basic information to get started. Obviously, you will have to optimize the system for your specific application, but I think ours is relatively close.
Protein Extraction
At harvest, each construct was rinsed with cold phosphate buffered saline (PBS) and frozen at −80 C until lysis. The tissue was then disrupted by sonication in ice-cold lysis buffer [25 mM NaF, 0.5% Nonidet P-40, 1 mM EDTA, HALT phosphatase inhibitor cocktail (Pierce), 1 g/ml aprotinin, pepstatin, and leupeptin (Sigma) in PBS]. The soluble lysate was isolated by centrifugation, and the protein concentration was determined by bicinchoninic assay (Pierce).
Western Blot
20 g of total protein/well (or 5 L of cell lysate from monolayers/well) was separated by reducing SDS-PAGE, transferred to a nitrocellulose membrane (Whatman), and immunoblotted for beta-actin (Sigma #A5441), phosphorylated ERK (#9101), total ERK (#9102), phosphorylated p38 (#9211), or total p38 (#9212, all ERK and p38 primary antibodies were from Cell Signaling Technologies). Briefly, the blots were blocked for 1 hour (block was 5% dry milk, 0.1% Tween-20 in PBS), incubated 1 hour with primary antibody (1:2000 in block for ERK, 1:2000 in TBS-T (Tris buffered saline + 0.1% Tween-20) + 5% bovine serum albumin for p38), washed in TBS-T, incubated 1 hour in secondary antibody (1:2000 HRP-conjugated anti-rabbit IgG, Amersham, in block), washed in TBS-T and PBS, and developed by enhanced chemiluminescence. Films were digitized using ScanJet G4050 set to transparency mode (Hewlett Packard) and quantified for band density using ImageJ (NIH).
It's not explicitly mentioned in the protocol, but the samples should be boiled for 5 mins @ 95C prior to loading the gel. This will help reduce any cross-linked collagen that might otherwise prevent the gel from running smoothly.
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