Episode#12 Immunoprecipitation – Erin Hughes

Barkha Yadav-Samudrala: 0:06

Welcome everyone, to another episode of nerd RX podcast. And I'm your host Barkha. And today we are going to talk about immunoprecipitation. And to walk us through what immunoprecipitation is, we have Erin Hughes, welcome, Aaron to the show. Thanks so much for having me. It was really excited for this opportunity.

Erin Hughes: 0:29

Thank you so much for being here with us. So, before we go into the topic, why don't you introduce yourself to our listeners? Sure. So as she mentioned, my name is Erin and I have a bachelor's in science in biochemistry, molecular biology, from otter ag find University in central Ohio, very, very small campus, like 2000 people. So don't be surprised if you haven't heard of it. I immediately went into a Ph. D program under the molecular and cellular Biosciences umbrella program at Wake Forest University in North Carolina. That's where I'm currently a fourth year PhD candidate in the biochemistry and molecular biology program. And I have the pleasure of getting to study a cannabinoid receptor interacting protein or crypt one A and its interactions between the G alpha subunit of the heterotrimer is and trying to figure out exactly what's going on with cell signaling.

Barkha Yadav-Samudrala: 1:23

Okay, wow, that's great. So Erin, would you mind telling us why did you pick immunoprecipitation? And what got you interested in it?

Erin Hughes: 1:35

So immunoprecipitation is one of those tools that can be used pretty much for everyone. But specifically with what I'm looking at, it's really helpful, because it's going to let us look at protein protein interactions or protein interactions in general, whether it's between two proteins or between protein, DNA, RNA, etc. So from my project, when we're trying to figure out how this protein crip is interacting with other proteins, how's it going to be, you know, modulating cannabinoid receptor activity, and all of that it's been a really great tool, just because it's going to let us look between two different proteins. And we have immunoprecipitation versus co immunoprecipitation, which is looking for actual complex, which has also been very helpful.

Barkha Yadav-Samudrala: 2:31

Right, so why is immunoprecipitation important, and what is it actually used for?

Erin Hughes: 2:40

So it's really important, because it's gonna let you look for known or even just suspected protein interactions in vivo and in vitro. So you can use cellular lysates, from whatever cell line you want to use, whether it's a cell line that has endogenous levels of proteins, or if it's someone that you know, you've transfected in your protein.And because there are so many proteins within the body, within the cell, they're all doing very, very different things, looking to see how these are interacting is going to let us downstream, kind of modulate those interactions. And even for example, use it as a druggable target. So eventually, you know, looking at how does this interaction occur? And can we stop that protein protein interaction from happening? And maybe we can, you know, help with pain control, or any sort of otherhealth effects that you might want to go for?

Barkha Yadav-Samudrala: 3:43

Wow. So it sounds like you can use this technique in a lot of different projects, I mean, different types of experiments.

Erin Hughes: 3:53

It's super flexible. You can you know, it's use it for a lot of different things, a lot of different areas, pretty much as long as you have an antibody to protein of interest. And some beads, you're good to go.

Barkha Yadav-Samudrala: 4:07

Yeah, antibody, I think is one of the issue with a lot of things because sometimes you don't have a good antibody and you're stuck there.

Erin Hughes: 4:19

That's actually been one of my major problems recently, when I've been trying to do my immunoprecipitations for my project, because you have to make sure that that antibody that you have is actually going to be to an epitope that's going to let you bind to your protein of interest. So if you have a protein that that is, for example, binding to another protein or a complex on the N terminus, and then you have an antibody to the N terminus that's probably not going to be able to be used as well because you're going to be blocking your binding site of your complex. So that makes it a little bit more difficult sometimes and trying to get you know an antibody for commercial commercially available antibody for a protein is sometimes also very challenging. I'm currently waiting on a custom antibody coming from the Pol.

Barkha Yadav-Samudrala: 5:08

I am too waiting for an antibody I've been like it's not immunoprecipitation but I am working on my western blots and the CB one and CB two receptors are like making me cry. I'm like, I still have a good data for CB two. But CB one is, I'm trying so many antibodies from different places, but none of them have actually worked for me.

Erin Hughes: 5:32

So I can tell you the CB one antibody we use is from Everest. Biotech.

Barkha Yadav-Samudrala: 5:39

Oh, I'm going to talk more about that after this episode. Yeah. Yeah, CB what has been a problem for me. So hopefully this works. Thank you so much for the tip. So I think in terms of Western Blot, I, my issues are first obviously finding the right antibody, and then the figuring out the dilution. So do you have the same issues?

Erin Hughes: 6:07

So that is something that you know, it's kind of thought about, but mainly for immunoprecipitations. You add typically, one microgram of your antibody to protocol I have from previous graduate students is between 100 to 500 micrograms of your protein. So in my case, that's going to be total protein within the cell, because we're trying to look at this like endogenously within the system of the cell. Okay, so usually the, that's just a typical, I have tried for lately, I've been having problems getting the immunoprecipitations to work 100% of the time, so I've tried to changing around the amount of antibody used but typically one to five micrograms of antibody.

Barkha Yadav-Samudrala: 6:57

Okay. Okay, that is good that that is figured out. So let's talk about the steps itself. So why don't you give us an overview for the entire process of immunization? Just an overview, like how do you begin? And what do you expect? And what are the various steps involved?

Erin Hughes: 7:20

Sure. So typically, you're gonna start with, you know, a protein lysate, or a Pantone eight or something, basically, something from cells or even purified recombinant, and a protein that has your protein of interest. And if you're looking for a complex, then well, you want to make sure all your other proteins are also in there. So once you have that, you're going to want to make sure to do some sort of Bradford or a BCA, just something to get a rough estimate of how much protein you have in your solution diluted down as needed, or just make sure you don't have way too much protein compared to your antibody. So once you have that, you're going to add your antibody, you know, typically one microgram, which oftentimes is only like a microliter or two of your especially with commercially available antibodies based off of concentration. And so this is where the protocols can kind of differ based off of experiences. But in my experience, we have figured out that if you leave the protein plus antibody mixture in the refrigerator overnight rocking, you tend to get better binding. So I leave that solution in the fridge overnight. So at least 16 hours after 60 doesn't seem to make a difference if it goes a little bit longer than 16 hours, as long as it's in there long enough to for things to kind of reach equilibrium and give that antibody a chance to basically go sorting around and find exactly what protein you're going to be looking for. Then the next day I add we use aigrow speeds, which are protein Ag plus beads and those protein ag beads are going to bind to the antibody. I usually let that go for a couple of hours at room temperature and then that's when you can pull basically spin down in a centrifuge your beads which is going to have your beads attached to the antibody attached to your proteins. From there you can do western blot analysis and then stain for your proteins of interest. You can also use magnetic beads and I know a lot of people have had good success with that. That's actually my next change to my protocol to try and get things 100% working is to swap over to magnetic beads, which are supposed to be really helpful too.

Barkha Yadav-Samudrala: 9:38

So are these beads like secondary antibodies?

Erin Hughes: 9:46

They kind of are the protein A G is going to bind to the IgG and IGA of the antibody itself so the long and short chainsso in some ways it is Like a secondary just attached to it I grocer magnetic bead.

Barkha Yadav-Samudrala: 10:05

Oh, magnet magnetic bead. This is the first I've ever heard of Could you tell us a little more about that?

Erin Hughes: 10:11

Yeah, so they are, you know, instead of Iger us which is what I typically use because it's cheaper you have a they're basically a little tiny magnetic beads balls if you will that have the protein ag attached to them same as your acuris. And then you can use a super magnet to basically pull the beads off to the side so that you can basically pipette or loot off anything that you don't want in your final solution, because you're just looking for what stuck to your beads. So usually you're trying to get rid of the stuff that didn't, which is going to be your supernate. And in that case, so it's a really great way to because the beads are stuck, you know, so closely to the side of your tube to make sure that you're not, you know, pulling your beads out of solution too early or mixing you're bound. You know what stuck to the bead versus unbound or supernait. And they are more expensive, which is one thing that some people don't typically use them for. But my experience has been they're pretty awesome.

Barkha Yadav-Samudrala: 11:14

Oh, wow. So after you're done with the immunoprecipitation and you have your bid, you go for a second technique, like you mentioned Western Blot.

Erin Hughes: 11:27

Yeah.

Barkha Yadav-Samudrala: 11:28

So does that means so when I do western blot, I usually have my brain homogenates. And I do a BCA to quantify how much protein we have. And then I just run my Western Blot. So with this immunoprecipitation what I am getting an idea is that you are kind of purifying it, you're just extracting your protein of interest, right?

Erin Hughes: 11:54

Yeah, that's right. So it's like a just trying to get that one specific protein out of one, you know, out of a solution that has 1000s of proteins. And in my case, I'm technically doing a co IP, or I'm looking for a protein complex, because I'm trying to see what crypts interacting with specifically in our cells. So it's just a good way to kind of pull out your only protein plus whatever's hanging on to it versus Western where you have way more proteins show that

Barkha Yadav-Samudrala: 12:26

your western blots would look super clean?

Erin Hughes: 12:30

Yeah, typically they are you get a very strong band of interest for your proteins. Low background. Yeah, just big increases the signal actually quite a bit.

Barkha Yadav-Samudrala: 12:43

Right? Yeah, I would imagine. Wow, now I'm considering leaning bit more, because the bands we get are super faint. And we have a lot of background signals. So I think this is something that will help us so. So just out of curiosity, for example, if I am trying to isolate CB one protein, right, and I use an antibody in the immunoprecipitation. And then, for example, a magnetic bead and I isolate and put it on the western blot. So do you use the same primary antibody for immunoprecipitation and the Western Blot?

Erin Hughes: 13:27

So I have in the past, but that depending on your type of depends on if your antibody is covalently linked to your beat or not. Because Okay, usually when you're trying to get your protein, it to basically pop off of your feed, you are going to heat it or we're going to use low pH or just something and my experience has been our antibody then comes off to be it as well. And it ends up being run on the western and some people it doesn't bother because the heavy light chain can run at a molecular weight that's not going to interfere with data analysis. But in my case, what I'm looking for actually runs at the same molecular weight as the antibody. So typically, my primary antibody tried to use a different species, which makes it kind of interesting sometimes when you're looking for multiple proteins we recently have tried using a chicken antibody which actually works remarkably well.

Barkha Yadav-Samudrala: 14:30

Wow. Yeah, I haven't trained chicken the only antibodies I'm working with right now is mice and rabbit and I don't know like I'm trying to find out CB one they only have rabbits CB one everywhere.

Erin Hughes: 14:44

He luckily we have a company that has goat CB one which has been fantastic because rarely gives another you know, another species for us not to have to worry about cross contamination with.

Barkha Yadav-Samudrala: 14:53

Yeah. Oh, wow. That would be super nice for me as well. Well, thank you so much for that description. And I can see how you can utilize this technique in so many different ways. So what is CO immunoprecipitation, because when I was actually reading up something on immunoprecipitation, before our episode, I came across something called immunoprecipitation.

Erin Hughes: 15:17

So coimmunoprecipitation is when you're looking for a protein complex, so not just two proteins, but a whole bunch of them, if you will. So I can use my research as an example with crip. We're looking to see if it interacts with G alpha i, so the G alpha subunit of the heterotrimer. Now, when we immunoprecipitated, looking for Krypton beta is suggested that Crips also interacting with g beta. So we have to kind of consider if CO IP ng crip is going to get us, you know, both Chi Alpha and Beta, as well as potentially gamma and even looking to see if CB one isn't there. So PCoIP is going to be looking for your actual complex of proteins. Because we know a lot of times in cell signaling and in general cell processes, that proteins are not doing their whatever they're supposed to be doing on their own, it's often happening with some other protein in some other part of the cell.

Barkha Yadav-Samudrala: 16:23

Okay, and Has it ever happened to you that you're trying to do immunoprecipitation for one protein, but you end up getting something else?

Erin Hughes: 16:35

Yes. And that oftentimes causes a little bit of a ha moment, because you're sitting there looking at data that doesn't quite make sense. And I think, especially if you're western blotting, and then you think you, you know, think of your think of using a negative control, and you going to blot for a protein that you don't think should be in the immunoprecipitation. But it does come up has happened and kind of has led us to want to send our samples off for mass spec just to see how much is actually coming down or immunoprecipitations with grip.

Barkha Yadav-Samudrala: 17:13

Okay, do you have any alternative techniques that you can use instead of immunoprecipitation?

Erin Hughes: 17:23

Yeah, so because you're looking for a protein protein interactions or things like that, you can run a Bret or fret which is going to be looking for fluorescent signal one, two proteins get close together I am, it's just things I have heard that I should be doing for my committee. So I have not really looked to too much into those yet. And whether or not we're actually going to use them or have much information on them. What I do in parallel as well as I do cell imaging. So I plate my cells on coverslips, and then stain for crip plus whatever antibody, whether proteins I want to be looking for and look to see if there's colocalization under the microscope, and I use few other techniques as well. Typically, you can, I would say running in parallel a or Brett fret cell imaging. You could even take your immunoprecipitation and put it on, do mass spec with it after instead of western blotting just to get your exact molecular weight of whatever proteins are floating around and solution.

Barkha Yadav-Samudrala: 18:28

And is this technique user friendly? Like how long will it take for a completely new person to learn this?

Erin Hughes: 18:38

So I think it's pretty user friendly, because there's a lot of protocols out there. And even a lot of companies have, you know, their version of the protocol available. So there's typically a little bit of troubleshooting, but I would say it only took me probably a couple of weeks to get really, really familiar with the technique. Now obviously, there's always going to be troubleshooting with anything that you're doing inside. The troubleshooting flow takes a while. But I think it's pretty simple because it's you know, you're mixing beads plus a solution and doing a lesson or figurations. The centrifugation patients get on your nerves after a while. Yes, yes. Yeah, I think troubleshooting is the main part of all the experiments. And I think you've spent so much more time on troubleshooting than your actual results. It you just have to get right once. So, troubleshooting is a big thing everywhere I feel.

Barkha Yadav-Samudrala: 19:38

So let's talk about the advantages and disadvantages of this technique.

Erin Hughes: 19:44

Yeah, the advantages would be that it's very flexible, especially if you're using a just an aggro speed with fruity ag or a magnetic one with protein ag that's going to be able to bind to any sort of antibody that you have in solution is long as it has an IgG or IGA on it. So that means that if you're looking at a wide variety of different proteins or your project is very diverse, you can kind of mix and match mix and match your proteins of interest with whatever antibodies you want to use. Now, the disadvantages being like I said, sometimes your antibody not might not find the way that it needs to be, or humid or precipitation isn't working quite right, or you're just not seeing signal on your downstream, you know, imaging your qualifications. So there's just kind of pros and cons to it just like everything, but I think my main attraction to it was being that I can pretty much look for whatever protein with whatever antibody want to and it's great for, you know, working parallel back and forth. So if you're Muna precipitating for CB one, for example, and you find that it interacts with the G alphas, then you can also immunoprecipitate with the D alphas to see if you get CB one, so you can kind of work in reverse there.

Barkha Yadav-Samudrala: 21:09

Okay. So have there been cases where immunoprecipitation cannot work,

Erin Hughes: 21:19

I have actually found that least with my methods that certain detergents can block some interactions of proteins with the antibody, which is not ideal in you. Because a lot of cell lysis buffers actually have, you know, detergent in them. That's how you burst open cells. So, not case I've had a time or two where the IPS just didn't work, there was no signal and I couldn't figure it out for a while. Or, you know, your antibody doesn't bind to your protein like it's supposed to. That's the other case where I definitely wouldn't work.

Barkha Yadav-Samudrala: 21:58

Okay, and this can be used even with tissue homogenate. Right. And not just cell likes it?

Erin Hughes: 22:04

Yeah, I would imagine. So I have not tried that. I think it's something that my advisor and I have talked about briefly, we currently don't have any animals or any brains for us to work with. But I definitely would say that you'd be able to do it, I think as long as your proteins are, you know, available for the antibody to find and bind. I don't see why I wouldn't.

Barkha Yadav-Samudrala: 22:26

Okay, awesome. And what would be the cost associated with it? Like, is this a technique that any lab can set up easily? Or is it expensive?

Erin Hughes: 22:39

I would definitely say it's very low cost. Especially if you're a lab that already has cells or brains or tissue homogenate already up and running on your day to day basis. The egg, especially the Agros beads, instead, the magnetic ones can be kind of expensive. But if you're trying this initially just to see if it's a technique you want to use, the Uygurs beads are very, very low cost. Well, you know, science expensive to begin with, but they're low cost in the world of science. Especially, do you think Santa Cruz biotech has protein ag beads for like $90 per milliliter of them, and you don't use that many so. So that is another advantage? I would say it's easy to set up.

Barkha Yadav-Samudrala: 23:29

So I think my last question for you would be is there any interesting articles or protocols out there that I can link down in my description for our listeners to read up on?

Erin Hughes: 23:43

Yeah, so there was when I was really digging into coimmunoprecipitation and immunoprecipitation, I came across this methods and molecular biology paper from 2017. That's just very simply titled Protein Protein interactions co immunoprecipitation. And I thought it was a great read just because it talked about how you can use genetic like yeast to use hybrid and biochemical. So your co IP methods to basically looked to see what proteins are interacting together. And it gave a really good protocol and background. That was think one of the first papers I ended up

Barkha Yadav-Samudrala: 24:21

So well with this. I think we are going reading about. to end today's episode. Thank you, Erin so much for being here with us and explaining what immunoprecipitation is.

Erin Hughes: 24:27

Yeah, thanks so much for having me. It was great talking with you.

Barkha Yadav-Samudrala: 24:38

Likewise, and listeners. I will catch you next week for another episode on this podcast. And it meanwhile, if you wish to suggest if you have any suggestions for any technique, or if you would like to join me on podcast and discuss a technique, please email me at Barkha@nerdrxpodcast.com and remember it's good to be a nerd, bye.