You have your favorite protein in mind and are ready to set up some exciting experiments to show what it does and how it does it, when it is active, what other proteins it modifies, and how it affects your cells. There is one slight problem. You need to fish your favorite protein out from the rest of the cellular sea before you can address your research questions. Read on to learn what gear you need to reel-in “the big one” using immunoprecipitation!
How to Fish for Your Protein-Of-Interest
Immunoprecipitation (IP) is a fairly straightforward protein isolation technique consisting of 6 main steps:
1. Gently lyse your cells
2. Introduce an antibody specific to your favorite protein
3. Introduce a small bead that binds to the constant regions of the antibody
4. Isolate the protein/antibody/bead chain either by gravity or by capturing it in a column
5. Gently wash your protein/antibody to remove unspecifically bound proteins
6. Finally, elute your favorite protein!
Sounds simple, right? But of course, there are many variables to an IP that can make the difference between an empty hook, and landing you the “big one”. Read below to learn what you can use IP for, and what factors your need to consider when selecting your IP tackle.
Why You Should Fish With IP
Some fishermen fish for sport, others for food. Similarly, there are many reasons why you may want to perform an IP. Here are just a few reasons why an IP might be the right method for you, beyond just the straightforward question “is my protein in there?”:
Determine Relative Abundance
Many proteins, particularly housekeeping proteins, DNA repair proteins, transcription- and translation- related proteins, are present in cells almost all of the time. However, if your protein’s abundance fluctuates – perhaps during the cell cycle or after a specific treatment – IP is a great way to show the “before and after” difference in its abundance.
Determine Protein Modifications
Ubiquitination, phosphorylation, sumoylation, and more! All of these post-translational modifications may occur to your favorite protein, providing clues about its function and/or its regulation. If you’re fortunate, you can purchase IP antibodies specific to modifications on your favorite protein and land two fish at once! If, however, such an antibody does not exist for your favorite protein in its post-translationally modified state, don’t worry – you can still do an IP to fish out your protein and determine its modifications otherwise. However, this approach will require an extra step in your protocol. You will need to 1) use an antibody specific to your protein-of-interest or the modification of interest, and 2) follow this up with a western blot to show that a specific modification occurred on your protein-of-interest at a specific time.
Measure Protein Depletion
Sometime, you may want to observe what happens to a cellular process in the absence of your protein-of-interest. IP can you let you do this by allowing you to deplete your lysate of a single protein. This allows you to see what happens when that protein is absent, or substituted by a mutant protein without having to clone the native protein.
Assess Protein Interactions
Few proteins function in isolation. And just like a healthy pond ecosystem, the cellular environment fosters complex and dynamic interactions between its residence. Luckily for you immunoprecipitation is an excellent way to not only fish for your favorite protein, but also catch your protein interacting with other cellular components. Using IP to assess protein interactions, however, does involve adding some extra steps to the basic IP protocol. Two common IP based methods to asses protein interactions are:
1. Co-Immunoprecipitation (Co-IP)
With Co-IP, your goal is to pull down your favorite protein and any proteins directly interacting with it. To do this you need to use gentle lysing, washing, and elution methods to preserve any protein/protein interactions. To learn more about this method, see our earlier articles on performing Co-IP and analyzing Co-IP results. However, it doesn’t stop there, because although Co-IPs are great to identify potential binding partners, interactions should be properly confirmed to eliminate building entire research stories based on proteins that simply stick together under the conditions of a Co-IP. Mutational analysis is one way to map binding sites and become more confident in an interaction, but for more specific information you may consider using a surface plasmon resonance (SPR) application.
SPR applications can confirm protein:protein interactions and provide quantitative analysis of the affinity, thermodynamics (through the use of SPR biosensors) and kinetics of these interactions in real time. If you are interested in studying interactions between immune receptors and their ligands, you may benefit from performing a biosensor assay. These assays follow SPR principles and allow you to measure association and dissociation rates between ligands and their specific antigen receptors. SPR has actually become the gold standard for kinetic and affinity determination, and forms the basis for most colorimetric biosensor chip applications.
2. Chromatin Immunoprecipitation (ChIP)
In this method your goal is to demonstrate DNA/protein interactions. To perform ChIP you 1) cross-link your lysate with formaldehyde or UV light to stabilize any DNA/protein interactions, 2) sonicate your lysate to fragment your DNA, 3) pull down your protein/DNA complexes with immunoprecipitation, 4) reverse your DNA/protein cross-links, and 5) analyze the immunoprecipitated DNA fragments by southern blot, PCR or sequencing to identify where your favorite protein interacted with DNA or a DNA/protein complex.
How to Fish With IP: Choosing Your Tackle
The Hook (a.k.a the Antibody):
The first consideration when designing your IP experiment is what antibody you should use. There are dozens of antibody companies who offer thousands of antibodies against individual proteins. So role up your sleeves, hit the net, and start searching through some catalogues.
Whenever possible try to find monoclonal antibodies that have a good track record in immunoprecipitation protocols. These antibodies are your best bet, because the manufacturer has done all the troubleshooting for you. They have already perfected the buffers, washes, and elution for your specific protein/antibody IP. So if you buy one of these proven antibodies all you need to do is replicate their experiments. Also, if you cannot find the manufacturer’s IP protocol, don’t hesitate to call them. They really can be very helpful!
The Reel (a.k.a. the Bead):
After your antibody recognizes your protein, you need to introduce a bead to recognize your antibody. This bead will act like a reel pulling in a hooked fish and physically pull down your protein/antibody complex. Beads are made of agarose or small magnetic beads embedded with either protein A or G. Protein A and G are both specialized bacteria proteins that bind antibodies. Whether you should use protein A or G will depend what species your antibody was raised in. So check the affinity charts associated with these proteins to pick the right beads!
The Pond (a.k.a. the Lysate)
Your fishing hole is your cell lysate. You can increase your chances of catching the “big one” by treating your lysate with care. Gentle lysis methods with mild detergents (e.g. NP-40, Triton X-100) are great if you’re looking for native, biologically active proteins. But harsher lysis buffers, such as RIPA, are often needed if you want to look for overall abundance or linearized proteins. Be ready to experiment with various lysis buffers and conditions to get the perfect fishing pool out of your lysate.
So, are you ready to catch the “big one”?
Originally published in 2014. Updated and republished in May 2017.Image Credit: Nina Hale
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