I´m new in this forum and my English isn´t good.
We are characterizing collagen typ I most of the time. I hope here are some collagen specialists?
OK, now I tell you my problem:
I have a 1% collagen dispersion (in 0,5M acetic acid) and I do a Lämmli SDS-PAGE with pre-cast Gels from SERVA.
I take Neutral Gels pH 7,4 and use the Lämmli-Buffer with 2-Mercaptoethanol.I boil the probes for 5 Minutes at 95°C. After that I centrifuge and start my Gel. Up to here it works but I am not really satisfied with my results, I often get a big trimer band at 300 kDa and the alpha 1 and 2 bands at about 120 kDa are very weak.
Then I Blot my Gel to Nitrocellulose and make a control dye with Ponceau S. Here I see my expected bands and much smear. Maybe other Proteins from my crude extract, (the collagen isn´t 100% clean, there are some unknown proteins in it)
Up to here it worked, it could be optimized aber I have at least the protein of my interest, but then the immunoblotting starts.
Here I used a polyclonal rabbit anti bovine collagen I antibody from AbD Serotec as first and a chicken anti rabbit IgG AP labeled as secondary antibody. And I have massive background, which comes not from the second ab. (I tested without first one)
I know that a polyclonal primary isn´t easy to use, I have offered a monoclonal collagen I antibody as well, but I thought it would be nicer with a bovine one. Especially for a later validation it would be nice if it works with this one.
I have tried several things to optimize but nothing works,
I used for Blocking 5% milk powder, 1 h at RT and parallel Blocking sol. from Candor
I make my antibody solutions normal in TBS-0,1% Tween 20 and 0,1% BSA and I tried versus Low cross buffer from Candor but it doesn´t help.
I detect with NBT/BCIP.
My control collagen from Sigma works ok, but my collagen extraction is totally pink. So I assume that the first antibody binds to the unknown and other proteins from cow.
I forget to tell that I have not tried a Native Page yet. Here I have a blot problem, but this is another topic. Maybe the antibody works there nicer but AbD Serotec has no information about this.
I can try PVDF or incubate at 4°C, but I have no hope that something will change.
Maybe someone else has more experience in that?
Edited by witchita, 30 December 2010 - 03:04 AM.