What collagen yre you trying to analyze? What I mention below works for type I collagen.

Are you doing a pepsin digest of your sample? Depending on what collagen you are trying to analyze this may be heavily crosslinked.

Before we analyze type I collagen from tissues or secreted type I collagen from cell culture supernatant, we digest the collagen with pepsin (0.1 to 1 mg/ml final concentration) in 0.1 N acetic acid, at least overnight at 4 deg C. We load this collagen, after adding Lämmli buffer, onto an SDS-PAGE (7.5% or 10%). Typically we can detect the alpha1- and the alpha2 band of type I collagen very nicely, but that depends also on the sample.

You may want to do a Western Blot and see whether you actually have the collagen of interest in your sample?

Hope this helped.

Best,

Claudia

Hi

What type of collagen are you extracting? As you are saying that you have digested it? After digestion, we get the peptides that have low molecular weight (3-6 KDa) where SDS Electrophoresis can detect proteins from 1-100 KDa. But What I suggest, instead of chemical digestion, you can try enzymatic digestion (as mentioned by Claudia Alma Staab-Weijnitz ). Further, instead of opting for Coomassie blue staining, try silver staining which is a sensitive method for detecting small conc. Hope that would help. Good Luck.

You can check these links:Article Hydrolyzed Collagen—Sources and Applications

Article Laemmli-SDS-PAGE