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public_enemee 09-08-2008 04:22 AM

primary vs secondary antibody

I'm actually confused about something. I just started using Western blot and usually refer to my seniors about the protocol. The problem is, one of my seniors only use primary antibody to bind with the protein (N protein). How about secondary antibody?Is that unnecessary?

For my project,I have to detect his-tag connected to the protein to make sure the his-tag is still connected to my protein after harsh cell disruption. Should I use primary antibody only (anti-his antibody) or secondary antibody also? If I should use secondary antibody, can i use anti-mouse to bind with anti-his antibody?

I really appreciate if somebody can explain this.

totoro 10-31-2008 06:01 AM

Re: primary vs secondary antibody

Try to ask your senior if the primary antibody is already conjugated to enzyme, either Horseradish Peroxidase or Alkaline Phosphatase. If yes, that's probably why he doesn't use secondary antibody.

This is a simple chart explaining how western blot works
his-tagged protein < anti-his antibody (primary Ab) < secondary Ab conjugated with horseradish peroxidase ---> the enzyme will react with fluorescence agent or chemiluminescence agent and generate light, so we could visualize it.

Here are the two advantages of using secondary antibody in western blot:
a. Increasing the signal
a lot of secondary antibodies can bind to various parts of the primary antibody. so compare which one is brighter: one primary Ab conjugated with enzyme vs. five secondary Ab conjugated with enzyme?

b. Save time and money
Imagine you have different kinds of primary Ab, all produced from mouse:
anti-His, anti-X, anti-Y, anti-Z,
to be able to generate the signal, you can either
i. conjugate them one by one with HRP enzymes
ii. you use anti-mouse Ab (=secondary Ab) that's already conjugated to enzyme. secondary Ab is also pretty cheap in general.

Hope that helps =)

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