Pull-down assays (co-IPs (co-immunoprecipitations), etc) - what, how, & what to look for

A “pull-down” experiment is where we capture (usually bind to beads) a molecule of interest (often a protein) from a mixture and see what it’s bound to (often via western blot or mass-spectrometry). One of the most common forms of a pull-down is a co-immunoprecipitation (usually just called a co-IP). As the “immuno” part of the name suggests, in a co-IP we’re using antibodies to do that capture step. But other forms of pull-downs are basically the same, you’re just binding through non-antibody-based interactions with some unique thing about the molecule of interest, such as an affinity tag (I will get into this later).

blog: https://bit.ly/pulldowns

Speaking of that “molecule of interest,” it is frequently referred to as the “bait” which is a lot shorter to write than “molecule of interest” so I’m going to go with that! We’re interested in seeing what “prey” that bait is bound to, so we need to:
1. capture the bait (and, indirectly, whatever it’s bound to)
2. Wash off non-bound stuff (under conditions harsh enough to remove the loosely-associated stuff (the wanna-be prey) but gentle enough to preserve the stronger interactions (the true prey))
3. Release the bait/prey complex
4. Figure out what’s there

Let’s go step by step…

1. The capture

The key things here are that:
we only want to capture the true bait. So we need our capture method to be very specific
We want to capture as much of the bait as possible, so we want this binding to be strong (high affinity)
We need to actually be able to isolate the bait, not just bind it to something
That’s all still talking about co-IPs (or plain-old IPs where you’re not trying to see what the bait is bound to just want to isolate the bait). But other pull-down strategies use non-antibody routes. They still rely on binding to a unique thing, which is often some sort of label you put on - it could be something like biotinylation (which can be captured with streptavidin and is a strategy sometimes used to capture labeled RNA or DNA) or an affinity tag like GST (which can be captured with glutathione) that you add onto the end of the bait through genetic engineering like with the epitope tags.

The genetic engineering is sometimes done in the endogenous context (e.g. using CRISPR to add the tag-encoding DNA onto the bait gene in place). Or it can be done on a copy of the bait in a plasmid that is transfected into (stuck into) cells (with or without knocking out the endogenous bait).

Whether it’s an antibody or glutathione or whatever you’re binding to, you need to be able to isolate it from everything else that’s in the mixture. And this part is usually the “easiest” these days thanks to magnetic beads. Basically these beads are attached (permanently (covalently) or temporarily (non-covalently)) to the thing you’re using for the capture (e.g the antibody). And you can use a magnetic rack to suck the beads to the side of a tube in order to remove the rest of stuff.

Alternatively, you can use non-magnetic beads, often agarose, and isolate them by centrifuging gently to pellet them out (hopefully without disrupting bait/prey interactions).

Either way, the capturing thing needs to be attached to the beads. If you’re using a common epitope like HA or FLAG, you can buy already-linked beads. Similarly for things like streptavadin and glutathione beads. But if you are using a different antibody, such as one against a native protein, you’ll probably have to do the attaching yourself. A common way that this is done is by using Protein A or G coated beads. These proteins are bacterial proteins that bind the genetic part of antibodies. So they will bind any antibody, but non-covalently, so you may have to worry about the antibodies coming off the beads when you try to remove your bait if your conditions are too harsh.

A note to know about this too is that the antibodies have light and heavy chains that will show up ~25 & ~50 kDa on an SDS-PAGE gel which could get in the way of your analysis (especially if you try to do a western with a probe against the species it comes from - avoid that if at all possible!)

So, we’ve somehow got the beads bound to the capturer. And we just need to capture our bait (well, actually first we wash the beads a couple times to get them adjusted to the conditions we want to bind in). After that, it’s capture time! Often we’re pulling the bait down from a lysate (broken-open cells) to see what was interacting with what inside of the cells (do they bind). But you can also use mixtures of purified components to see if they bind (can they bind). Sometimes people do a sort of hybrid where they add a purified, labeled bait to lysate. This is less physiologically relevant than having the protein expressed in cells, but it gets around the need for manipulating DNA in the cells.

finished in comments