Collagen I and V western blot - Blue Native PAGE - Blue Native PAGE (Jan/14/2008 )
Hello everybody!
I am new in the BioForum, and I really found it very interesting and helpful.
I wonder whether you can help me with my experiments. I am isolating total proteins from human aorta and I am trying to do western blot for collagen I and V. I have 2 antibodies from Abcam (ab292 COL-I and ab7046 COL-V) which work on native collagens according to the instructions from Abcam. So, I tried to do blue native PAGE. I used 4-16% native gels from Invitrogen and the protocol that they provide. I used DDM 2% as a detergent for my samples and I loaded 40ug total proteins in each well. I tranferred the gel to PVDF membrane, I used the rabbit anti-human primary antibodies and a mouse anti-rabbit HRP secondary (Santa Cruz). The expected molecular weights of native COL-I and COL-V is ~400kDa. I used ECL for detection of the bands. I am getting results, but my bands are on the top of the gel (MW >1200kDa). In fact they don't run in the gel at all. I have repeated the experiment 2-3 times, by using another reagent for total protein isolation (T-PER solution from Pierce), but again I am getting the same results. I have seen that somebody used tris-acetate gels 4-8% for collagen PAGE. What do you think? Do I need to switch to tris-acetate? But again I think that I have to proceed with a Native protocol. Is there any way to be able to run these collagens into the gel? Another detergent to increase solubilization? Any comment is welcome.
Thank you in advance.
you can try the tris-acetate buffer system. it is still a native (nondenaturing) gel.
Thanks mdfenko! So, you mean to use a tris-acetate gel and its native conditions? The rationale for this is that may increase the solubilization of collagens?
yes, to increase or maintain solubility. what does it say in the paper with the protocol?
the point is to use what you know works. no need to reinvent the wheel.
Maybe a gradient gel 4-20% is a better choice for large proteins? Pre-casted gels are readily available.
a gradient gel would be good if he/she were looking at a range of molecular weights but he/she is looking at two high molecular weight proteins of similar mw. in order to separate them a straight low percentage gel would be best (maybe some charge differences will allow them to be separated).
Thank you guys for your help. I think that it is time to proceed with the tris-acetate gels. There are two types from Invitrogen: 3-8% and 7%, so the best solution is the first one. Actually, I don't have a paper for collagen (fibrillar) PAGE, that's why I am asking these questions. The problem is to force the proteins to run into the gel, which was not possible with the blue native PAGE (My antibodies seem to work good though).
Hi everyone, just register to this wonderful support system so we could all advance in research. I am trying to do a western on type 1 and type 3 collagen from mice heart. I have not started to do the extraction protocol because I’m a little puzzle where to start. Some protocol used 2% SDS, formic acid and CNBr to solubilize collagen but I do not know if this will denature the collagen to the point that my polyclonal antibodies will not detect it. If anyone has a collagen extraction protocol for tissue samples or know of good references, please share. What is the pro and cons of collagen detection of its native vs. denatured form? After getting some help with the extraction of collagen I could probably take some of the recommendations posted of running the collagen onto a gel and do the western-blot transfer. Any help will be appreciated.
Thanks