Western blot bovine collagen typ I problem - (Dec/30/2010 )
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?
are you seeing general background or just in the lanes?
if it is general background then you may need to change your blocking agent. with a chicken secondary antibody i would use 2-5% normal chicken serum (ncs) to block the membrane. i would also add 1-2% ncs to the antibody solutions instead of 0.1% bsa (i would have used 1-2% bsa).
if the background is just in the lanes then the primary antibody is also picking up fragments and isomers of the collagen. the antibody also may not be as specific as you require. you may have to try a different antibody.
Thank you Mdfenko,
I have only background in the lanes with bovine collagen, the control probe with chicken collagen typ II is negative.
I guess that the first antibody makes my background, therefore I offer a monoclonal first antibody and another second antibody.
The hint with chicken serum is good, maybe I´ll try.
I would like to have the protocol for collagen SDS-PAGE. The collagen obtained by tendon dissolution with acetic acid. I am not sure whether the acetic acid will hinder the protein quantification by BCA assay and further SDS-PAGE. Do I need to dialyse the collagen into any other solvent before running BCA Assay and SDS-PAGE? Thanks