Pull-Down Assay Protocol

Protein interactions reveal a lot about how proteins and cells function under different conditions. One tool that allows us to look at direct protein interactions is called a pull-down assay. A pull down assay utilizes a bait protein bound to beads in a column to catch protein binding partners. This technique can be used to verify a predicted protein interaction via Western blot or identify novel protein interactions using a total protein stain.
  • a reversible protein modification where a phosphate group is covalently attached to a protein. The phosphate group regulates the function of the protein
  • the structural units that form proteins
  • a method used to purify a biological material from a mixture utilizing the physical binding/interaction of two substances (eg. antibody and antigen, receptor and ligand, protein and nucleic acid, etc.)
  • a type of affinity chromatography where one protein is purified from a mixture (prey) because if it’s affinity for an immobilized binding partner (bait protein)
  • the breaking down of a cell due to damage of the outer cellular membrane
  • a solution with a designated pH that is used to remove impurities or excess substances from a reaction or mixture (eg. a wash buffer removes excess protein from a pull-down assay, and it removes it removes salt or cellular debris from nucleic acid extractions)
  • a liquid (which pay or may not contain particles such as protein, salts, or other molecules) that has passed through a column
  • the process of removing one substance from another, usually by addition of a solvent
  • a technique used to detect specific proteins from a larger mixture
  • a microporous substance which binds proteins
  • a large, “Y” shaped protein that binds directly to one protein or antigen with high specificity
  • a large, “Y” shaped protein that binds directly to the Fc region of a primary antibody
  • a set of protein standards used to approximate the size of experimental protein samples run during gel electrophoresis
  • the transmission of cellular signals from the external environment to/through the internal environment of the cell

The cells in our bodies are working around the clock to perform vital functions that help keep us alive. Cells use proteins to carry out these important functions, so scientists often want to know about the abundance of different proteins within a cell under different conditions, how proteins are made and degraded, and which proteins are bound to each other. Proteins are constantly interacting with each other by adding or removing phosphate groups, forming complexes, or even cleaving off amino acid subunits. These interactions can reveal a lot about how proteins and cells function under different conditions, but since we can’t just look inside a cell and see how proteins bind to each other, we need a better way of understanding protein interactions.

One tool that allows us to look at direct protein interactions is a type of affinity chromatography called a pull-down assay. This technique is sort of like going on a fishing trip where you use specific bait to catch the specific fish you’re looking for, only in this case, we use a protein as bait to catch its binding partner. Let’s imagine that we’re researchers trying to understand how cancer cells evade our body's defenses. We have identified a protein that is correlated with cancer (protein A), and we hypothesize that it binds to protein B which is a protein involved in a signaling cascade. Verifying whether these two proteins interact will give us more insight into the inner workings of this type of cancer cell. A pull-down assay would allow us to test this hypothesis and jumpstart our investigation into the function of protein A. It might sound a bit bizarre to think about a pull-down assay as a fishing expedition for proteins, so let's take a look at how this technique works.

To do a pull-down assay, we use something called a column, which is basically a little tube with lots of tiny beads inside. These beads allow us to attach our bait protein to the column so it stays in place and can catch our prey protein. You could think of the column beads sort of like a dock. A person can stand on the dock and cast a fishing line with a piece of bait into the water to try and catch a fish. We’re going to do something very similar in our pull-down assay.

Let's go back to our cancer investigation. We’re interested in determining whether protein A binds to protein B inside a cell, so I’ll make protein A my bait, so I can see if they actually do bind to each other. If the two proteins interact with each other in the cell, we should be able to reproduce that interaction in the column. The first step in our little fishing expedition is to attach our bait (protein A) to the beads in the column. To actually make the protein stick to the beads, I have to give it a little tag so that it can bind. I won’t get into the specifics of how this tag is added, but the important thing to know is that it allows our bait protein to remain immobilized on the column. Once the bait is bound to the column, I can fish out the prey protein from the large population of other proteins in the cell. In actuality, a bait protein might have more than one prey that it binds to, but right now we’re only interested in checking a specific binding partner for protein A.

Basically, what I do is take cancer cells, burst them open (or lyse them) so that all the proteins come out. Then, I run that protein solution through my column. When a protein that normally binds to Protein A is in the vicinity, the two proteins should bind. Remember that Protein A is the bait protein and is stuck to the beads in the column, so any protein that binds to Protein A will be stuck in the column as well. Then, I add a wash buffer to the column which helps wash away any proteins that didn’t bind protein A. The stuff that comes out of the column during this wash step is called the flow-through, and it gets collected in a little tube and usually discarded or used in a different experiment.

In parallel, we have to run an important control alongside our experimental sample that ensures any prey protein we capture is actually binding to the bait rather than binding non-specifically to the column. Proteins can be rather “sticky,” so we want to be sure that our prey protein isn’t binding to parts of the column instead of the bait. To control for this non-specific binding, what we do is run a second column in parallel with our experimental column, running exactly the same steps I just described but without the initial step of linking our bait protein to the column. By excluding the bait, the only things a protein could bind in this control column are elements of the column itself. So, we’ll eventually be able to compare the data we collect from the control column to the experimental column to exclude false positives from our final experimental analysis.

So, at this point in our pull-down assay, we’ve fished proteins that bind to Protein A out of the total pool of proteins and controlled for non-specific binding to the column, but we still can’t see these proteins by eye. That means we need a way to identify what we’ve caught. The first thing we have to do is get our proteins out of the column, and there are a few different ways to do that. One way to remove our proteins, or elute them, is to use a special solution to release all the proteins from the column, both bait and prey, so they can be collected in a new tube. Passing this solution through the column allows us to gather the released proteins in a collection tube for analysis.

To identify the proteins that have bound to bait protein, we’ll rely on our old friend the Western blot. We go through the same basic procedure of denaturing our proteins, running them on a gel, transferring the proteins to a membrane, and probing the membrane with antibodies, meaning we tag and visualize our proteins of interest - in this case, Proteins A and B.

Let's talk through the results we got for our cancer proteins. We know how to visualize proteins for a normal Western blot, but in this case, we need two different primary antibodies because we’re looking for two different proteins. After we’ve probed the membrane with those separate primary antibodies, we’ll have 2 different pictures. To make this analysis a little simpler, let's imagine that we superimpose those two images of our membrane on top of each other so we are able to visualize both proteins of interest at once.

Now that all the data is in one place, it will be simple to analyze the results. The first lane is the protein ladder that allows us to identify how large our proteins are. The second lane is our control sample where we used the column that didn’t have any bait protein stuck to it. An empty lane means that protein B didn’t bind randomly to other parts of the column. Remember that we didn’t attach protein A to the column in our control, so there shouldn’t have been anything for protein B to grab onto as it moved through the column. The only way protein B could have remained in the column during our wash step was if it was bound to the beads and since lane two is empty, we can be sure that this didn’t happen. This gives us confidence that any results in the experimental column are biologically relevant to what’s happening in the cell. The third lane is our experimental sample where we used protein A as bait. If we see two bands, that means both proteins A and B came out of the column when we did that final elution step, so that tells us that protein A successfully pulled down protein B with it. In this case, since we know the sizes of our two proteins of interest, we can compare the bands to our protein ladder to confirm that we obtained the expected sizes.

This result supports our initial hypothesis that proteins A and B bind to each other in cancer cells. Since we know that protein B is involved in signal transduction, this gives us a better picture of how this specific pathway might contribute to cancer. This is a great use for a pull-down assay, but we could actually take it one step further and see what else we can discover about protein A.

I said before that it’s possible for proteins to have more than one binding partner, so we could use this same assay to see if protein A binds to other proteins besides protein B. We would go through the same procedure of anchoring protein A to the column, adding our pool of proteins from the cancer cell, washing unbound proteins from the column, and eluting the bound proteins. Although we may want to do a Western blot to verify the presence of Protein A, we don’t know the identity of any additional binding partners so we can’t use any other specific antibodies for our analysis. Antibodies are helpful when you know exactly what you’re looking for, but here we need to use a stain that generally identifies all proteins that came out of the column with protein A.

If we take a look at these results, we again see our protein ladder in lane one and nothing in lane two which validates the specificity of the rest of the results, but now we see three bands in lane three. We can see the same band that corresponds to the size of protein A and protein B (which we know from our previous experiment) but now we see that there is a third band as well. This means that protein A actually binds another protein besides protein B. This information doesn’t tell us what protein that is, but it could spark a new investigation into what the mysterious third protein could be which could lead to more discoveries about how this cell has become cancerous.

This is just one example of how a pull-down assay can be used to study a disease. This technique can be used to identify novel protein interactions or assess how proteins bind under different conditions for any number of diseases or simply provide information that can offer a better picture of what’s happening in the inner world of cells.

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