I would first check if your antibody has been tested for western blotting from the manufacturer. Does it work in other methodologies, like immunohistology? Also I'm sure you've tried, but maybe experimenting with a far range of concentrations to determine if the concentration just isn't high enough. Also check the transcriptome or genome to make sure the immunogen is well conserved in whatever species you are testing in. After that, I would start thinking about other alternatives.

 hi mr.suriano

thanks for the answer.but the calf skin collagen type 1 that i use,has a trading mark(sigma) and i use it just as standard marker to compare with my collagen sample extracted from sea cucumber.how can i check the transcriptome or genome of calf collagen protein.

Hello, I actually came across with the same problem. I was trying to use calf skin collagen solution from Sigma (I don't know if it was the same one, but if it was it came diluted in acetic acid, at a concentration of 0.1%) as a comparation to my collagen samples.

At first the Calf skin collagen lane showed no band at all, and that might have been because of the pH of the sigma solution. If you noted, probably after adding the "charge buffer" the sample didn't turned blue like the others, instead it turned yellow.

What I did was: After adding the charge buffer to my sample, I used NaOH to neutralize it. Of course I used just some uL because I was afraid of diluting the sample too much. The sample inmediately turned light blue, a little more weak than the other ones. After running the SDS PAGE I noticed an improvement, and the apparition of bands. Of course they were weak because no matter how little of the NaOH solution I used, that only meant that I was obtaining a very diluted sample of calf skin collagen.

Maybe you can start playing with the volume of the base solution to neutralize the pH, so you can see some bands.

I hope it was helpfull.