Hello everybody! I was doing western blotting to assess Collagen 1 in my rat samples. I did not have any problems with the running or the transfer. However, I had a lot of different bands with the antibody I was using. So, we decided to test a new antibody for Collagen 1. I looked for such antibody and I didn't find any antibody that recognize the denatured form of the protein. Because of this, I would like to know what changes on the WB protocol to identify a native protein. Thank you very much for your help!
Hi,
First, it would help greatly if you would describe the exact protocol and antibodies your are using, therwise my answer is reliant on speculation. Especially if you are using a polyclonal as your detection antibody.
So here speculation: in short it depends partly on what type of SDS-PAGE you are running before the WB. If your are running the PAGE in denaturing conditions or you prep your sample in standard fashion (SDS, B-mercaptoethanol), your protein is naturally going to be in non-native condition. Based on your description my guess would be that your native 360 kDa collagen may have been degraded partially to smaller constituents.
The transfer to nitrocellulose membrane may also affect the native conformation of some proteins but should not normally result in breakdown of protein complexes. the effect is similar if you would coat your sample on a microtiter plate. New epitopes will open up for sure.
You could try to look for collagen 1 related literature and check what protocols are used to analyse it on WB (denatured and native), and what type of band-profile you could expect.
Best of luck!
Hi,
I completely agree Juha Kotimaa, you should specify the clonality of Antibodies and conditions used for your blot development. Also, you have to optimize different conditions like incubation, washing, transfer and evel development for minimizing multiple bands in blots.
Best of luck...
Don't use SDS, B-mercaptoethanol, maintain cold condition, standardize different concentration of sample protein, overrun if your antibody have more molecular weight (>250kb), use monoclonal antibody, apart from these you follow the above suggestion by Mr. Juha Kotimaa.
Good luck
Like stated above, native, also called reduced or non-denaturing, is when no SDS is added to the migrating buffer BUT also means to NOT heat the samples. When you don't add B-mercaptoethanol, that is calling it oxidized and non-reducing.
A good written protocol by Abcam that has a chart explaining this is attached. Hope this helps!
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