Why we use secondary antibodies (indirect detection method) - in western blots, etc.

Описание: Why use secondary antibodies? Signal amplification (so greater sensitivity) & cost savings!…

blog form: https://bit.ly/secondary_antibody

In the lab, we often use antibodies as “probes” - molecules that let us check for the presence of other molecules. Antibodies are little proteins that recognize & bind specifically to specific parts of other things, with the “other thing” being called an ANTIGEN and the “specific parts” being epitopes. For example, in a western blot, where you look for specific proteins by separating proteins in a mixture by size using gel electrophoresis, then transferring the proteins onto a membrane, and probing it, the antigen is a protein you’re looking for and the epitope is a specific site on that protein.

The primary antibody recognizes your specific protein and binds it, so you get your specificity for the thing you’re looking for - yay! But it (at least usually) doesn’t have anything “seeable” about it. In order to see where it’s bound, we bring in a labeled secondary antibody (after washing off non-bound primary antibody).

The secondary antibody recognizes your primary antibody and has some “detectable” quality. Commonly this involves them being conjugated (attached) to a molecule such as:
a fluorophore so it will emit light if you shine light of its favorite wavelength at it OR
an enzyme like horse radish peroxidase (HRP) that will convert an uncolored compound you add to a colored compound (chromogenic method) or light (chemiluminescence)
this strategy allows for greater sensitivity than the fluorescent method, just takes a (really quick) extra step

It works because antibodies have a unique part that recognizes some antigen and a “generic adapter part” - but that generic adapter part’s only generic for the particular animal that made it (i.e. the adapter part’s slightly different in mice & rats). So you can use things like “goat anti-rat” which is a secondary antibody that uses its unique part to recognize the generic adapter part of a primary antibody made by a rat (and if that rat antibody is using its unique part to bind your protein…)

We use this 2-tiered strategy (sometimes called “indirect detection”) so that we don’t need “fancy” (detectable) antibodies for every single protein we want to look for. Primary antibodies are expensive enough as is! And since different people will want different ones, and each person will want multiple ones, it doesn’t make financial sense for companies to sell labeled primary antibodies*

Primary antibodies are only made in a few animals (often mouse or rabbit), so companies only need to make and sell a few different secondary antibodies that will work with tons and tons of primary antibodies. And the companies will conjugate those to different types of labels and stuff and still be able to sell lots, so you get a bunch of options to choose from.

*labeling the primaries doesn’t make financial sense except in the case of “housekeeping proteins,” which are proteins that are expressed at fairly constant levels across cell types & conditions and therefore allow for normalization to control for different amounts of sample loaded per lane. Much more on this here: blog form: https://bit.ly/housekeepinggenes ; YouTube:    • Housekeeping genes/proteins, internal refe...   & https://bit.ly/western_reprobe ; YouTube:    • Probing a western blot membrane for multip...  

But since lots of people use the same housekeeping proteins, companies can profit based on selling lots even if they sell them way cheaper, and since housekeeping proteins are typically expressed (made) at highish levels, you don’t need awesome sensitivity - which brings me to my next point…

Secondary antibodies increase sensitivity because multiple secondary antibodies can bind each bound primary one so it amplifies the signal (similar to the way in which polyclonal antibodies (which are a mix of antibodies that bind different epitopes on the same antigen) can have greater sensitivity than monoclonals (which only have (lots of copies of) a single antibody). More on antibodies here: http://bit.ly/antibodytypesanduses

After you bind the secondary antibody, you wash off any that’s non-bound and then you can go visualize it, detecting the detectable using the detectable’s detection method (shine light at it, add substrate (the molecule the enzyme will modify), etc.

more on western blotting: http://bit.ly/westernblotworkflow ; YouTube:    • Western blots: the what's, why's, and whic...  
             
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com