Immunoblot (western) resolution vs electrophoresis resolution - (Dec/03/2005 )

Hi,

I did an SDS-PAGE electrophoresis for the proteins in an E. coli extract and colored them with Coomassie Brilliant Blue.

I also transferred the proteins to a PVDF membrane for immunoenzymatic reaction, to detect which proteins were recognized by a mouse that was previously immunized and had synthetised antibodies against.

My question is :

Why is the resolution (the bands' thickness) lower after transferring the proteins to a PVDF membrane and coloring with BCIP/NBT reaction? In other words, the gel, when colored with Coomassie, gives very thin bands, but the membrane gives thick bands.

Thanks.

JetW8

Edit : Also, ELISA vs immunoblot (western blot), how does sensitivity compare between both?

PS: i'm a student in Biomedical Sciences, not an expert!

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First off, I don't think resolution is the correct word. I know what you mean. To answer your immediate question, Westerns are more sensitive than Coomassie blue staining as you are able to amplify your signal TREMENDOUSLY w/a Western blot, but can't w/staining.

Silver staining is more sensitive than Coomassie btw.

As for band thickness the reason you see larger bands from the blot is because you have many antibodies binding to the protein. This amplifies your signals from many different points on the same protein. If your primary Ab is detected by a secondary (2 step amplification) your signal is increased even larger, as many secondary Abs may bind to single primary Ab.

Now w/Tyramide Signal Amplification (TSA, an NEN product) you can truly amplify your signal beyond belief (it also brings up heavy background often). TSA can really make an extremely faint or undectable signal appear. I believe dilutions of primary when used with TSA are often in excess of 1:100,000 even 1:500,000 I have heard.

Now what I don't know, is if fluorescence based scanners (Odyssyey by Li-Cor, or a Typhoon system) can amplify a weak signal better than TSA.

Hope that helps.

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