Questions about antibodies

[JFK90787]

OK, me am stupid. Can anyone help me? Nobody in my lab speaks English and wikipedia isn't helping.

1. Polyclonal antibodies just means a mixture of antibodies that bind to different epitopes on the same molecule. Right?

2. For immunohistochemistry, I understand the primary antibody is produced in a certain animal and binds to the antigen, and the secondary antibody binds to the primary antibody. Does the isotype of the antibodies ever matter for this, or is it just a matter of the correct animal? EG I'm using a primary antibody that is IgM and produced in mouse. Can the secondary antibody be something like IgG anti-mouse?

3. Why is there no such thing as fluorescent primary antibodies? Wouldn't this be easier?

4. Is part of the constant region specific for animals of a specific species?

Thank you to anyone who helps

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[Ariodant]

1, yes.
2, I personally try to match the isotype, because the Fc is different between different isotypes.
3, immunostaining aims to amplify the signal. One antigen is bound to many primary antibodies, while each primary in turn can bind many secondaries. Most proteins are not abundant enough to be seen with only primaries. Plus engineering fluorescent primaries against thousands of antigens is not as feasible as fluorescent secondaries against a limited number of hosts.
4, I believe so.

 

[xanthines]

1. Yes
2. Agree with ariodant. Match if you can, but not the end of the world if you can't. You'll have to figure out if this yields weird staining on a case-by-case basis.
3. There are lots of primary-labeled antibodies out there. We use them in flow cytometry all the time. Ariodant is correct that fluorescent primaries may not be strong enough for IHC, but this is antibody-sample dependent.
4. I'd say it's selective, rather than specific. You can get some cross-reactivity in certain cases. Although not strictly for this specific question, some companies make cross-adsorbed secondaries for minimal cross-reactivity across species. This is useful if you're double-staining using antibodies from two different host species.

 

[JFK90787]

Thanks for the help guys. I'm having fun in this lab but sometimes it's just a little intimidating asking questions about things that the people around here seem to expect me to already know