Protein Purification and Method Development Using PhyTip Columns
Protein Purification and Method Development Using PhyTip Columns

High Performance Immunoprecipitation

What is immunoprecipitation (IP)?

IP is an abbreviation for individual protein immunoprecipitation. Immunoprecipitation (IP) takes advantage of natural antibody antigen interactions to purify a particular protein from a sample of biological origin. The process uses a specific antibody to capture its counterpart antigen protein in sample types such as crude lysates of bacteria, plants, animal tissue or other various body fluids. This method enables protein isolation and concentration to aid in the study and assay of proteins of interest that would otherwise be difficult to detect in applications such as western blotting. IP can also help identify and interrogate protein-protein interactions.

Operationally in IP, a protein mixture or cell lysate containing the protein of interest is brought into contact or incubated with a specific antibody chosen for the purpose. Antibodies specific for given proteins are commercially available from a number of companies. The “immuno” in the term immunoprecipitation refers to the antibody selectivity and mechanism for capture of the protein. The “precipitation” portion of the term refers to the capture of the complex onto a solid support. There are different ways that IP can be performed to recover the protein of interest.

What are the different ways that a PhyTip columns can be used to purify a protein of interest in an IP experiment?

There are two basic antigen/antibody capture methods used in IP. The first method, called the indirect method, involves forming the antibody/antigen complex in solution prior to isolation.
After incubation, the complex is captured by passing it through a PhyTip Protein A or Protein G column. The antibody portion of the complex attaches to Protein A or G resin in the PhyTip tip, pulling the antigen along with the antibody. After capture, the complex is washed and then eluted off the column using a low pH buffer.

One of the advantages of forming the complex first and then capturing is the time, temperature, and chemical conditions for complex formation can be easily and finely controlled. In this way, complex formation is reliable, predictable and reproducible. For example, the complex formation can be performed in a refrigerator at 4oC for a specified time with specific reagent concentrations. However, formation of the complex relies on the diffusion of reagents to make contact. Mixing by sonication can be performed in some cases. Diffusion or mixing of this type is very slow sometimes requiring hours, or even overnight processing.

Formation of the complex is an equilibrium process therefore, the concentration of the antibody and/or antigen is a factor in how rapid and to what extent the complex can be formed. Because the reagents may be dilute in the indirect method, the driving force for complex formation can potentially be low. Both of these issues of diffusion and driving force can slow formation of the complex and reduce the amount of complex that is ultimately formed. Nevertheless, because of the tip concentrating effect, the high concentrations of the final product are eluted from the PhyTip column. Further information is available in the PhyNexus white paper “High Performance Immunoprecipitation Using the PhyNexus PhyTip System.”

The second method, called the direct method, involves loading the antibody directly onto a
Protein A or G PhyTip column by back and forth flow. The conditions can be optimized for high loading of the column while still using small amounts of antibody. After the “antibody column” is constructed, the column is placed in the sample and the antigen is captured from solution, again by using back and forth flow. There are several advantages of loading the antibody onto a Protein A or G column and then capturing the desired protein. First, a controlled, high concentration of antibody is introduced to the Protein A or G resin via active transport (back and forth flow) as opposed to diffusion. Active transport (actively flowing or bringing the reagents to the reaction site) decreases the time and increases the control of the antibody so that very little reagent is used in each experiment. In effect, the researcher is making a selective resin for the next step, the capture of the antigen. Because the antibody is captured on the resin in high concentration (relative to bulk solution) and the sample antigen is brought to the antibody via active transport (back and forth flow) the capture of the antigen is rapid, predictable and efficient. After capture, the complex is washed and then eluted in with low pH buffer as normal. This procedure is another variation of High Performance Immunoprecipitation (HPIP).

Why is IP by PhyTip called “High Performance Immunoprecipitation (HPIP)”?

High Performance IP is characterized primarily by speed and miniaturization with the following key attributes:
1) Fast; Active transport drives the equilibrium of the reaction and dramatically decreases the time required to obtain high purity targets.
2) Linear; Back and forth flow pushes the equilibrium reactions to completion. Reagent concentrations can be adjusted to shift reaction equilibrium and control speed of equilibrium.
The amount of antigen recovered is proportional to the amount of antigen in the sample.
3) Sensitive; Concentrations of the recovered protein are higher than by any other method.
4) Predictable: Again, column interactions are driven to completion. Conditions and time can be controlled. If using a PhyNexus MEA, experiments can be performed in a refrigerator giving temperature control to the process.
5) Because the method is controllable and predictable, any set of conditions developed by an operator in one laboratory can be transferred to another operator or even another laboratory.
6) Parallel operation; The process is controlled and can easily be performed under exactly the same conditions.
7) Cost effective; Small volumes of resin in the tips and active transport to load the antibody make effective use of costly antibody and reagents at no cost of performance.

You mentioned that PhyTip HPIP is cost effective by making effective use of the antibody.
How is this possible?

It may seem counterintuitive, but the best results are obtained by using the smallest column possible. The Tip Concentrating Effect described in this question and answer explains that the highest concentration of protein is recovered from the smallest column. . Because a small column can be used effectively, the amount of costly antibody needed to load the column is relatively small. We recommend the 20 μL bed column for HPIP. But if an antibody is more expensive than normal, it is probably worth trying out the 5 μL bed PhyTip column and comparing the results. In many cases, the 5 μL bed will perform very well. Another interesting aspect of using the smaller bed column is the reduction of nonspecific binding. Since the Protein A or Protein G column is small, it is possible to load up virtually every site on the column. Increasing the concentration of the antibody on the column reduces the possibility that other (nonspecific) contaminants bind to the column and are recovered with the antigen.

Can HPIP be performed without using different antibodies?

Literally thousands of different antibodies are now available from perhaps a dozen companies.
However, it still may be difficult or inconvenient to acquire an antibody that specifically targets a particular protein of interest. To simplify this, researchers will engineer antibody selective tags onto either the C- or N- terminal end of the protein of interest. The advantage is that the same tag can be used time and again on many different proteins and the researcher can use the same antibody. Examples of tags that can be engineered into proteins are the Green
Fluorescent Protein (GFP) tag, Glutathione-S-transferase (GST) tag and the FLAG-tag tag. There are several examples of an antibody that are selective for a particular tag e.g. GFP that can be purchased and bound or captured by a PhyTip column. While the use of a tag to enable pulldowns is convenient, the method may not be as biologically relevant. The tag itself may either obscure native interactions or may introduce new interactions.

What are GST tagged IP and HIS tagged HPIP?

This procedure is somewhat of a misnomer but should be mentioned here because of its simplicity and power. The technology, sometimes called a “pull-down” affinity purification technique, is similar to immunoprecipitation except that the antibody function is replaced by some other affinity system. In this case, the affinity system is either a GST-tagged protein that can be captured by glutathione agarose beads or a His-tagged protein that can be captured by Ni-IMAC beads. The recombinant tagged protein acts as the “bait” to capture a specific binding partner sometimes called the “prey.” The procedures and methods of use are the same as conventional IP. For example, the His-tagged bait protein is incubated with a cell lysate and the protein complex pair is captured on PhyTip IMAC column. After washing, the protein complex pair is eluted for analysis by gel, western blot, mass spectrometry, etc. Alternatively, the His-tagged protein can be loaded on a PhyTip column and nonspecific bound material washed away. Then the PhyTip column can be used to capture the protein of interest and recover the protein.

What is a false positive?

A false positive is a recovered protein that is not actually associated with the antibody baited protein. False positives can occur through exposure of the proteins in the sample to a high surface area, non-polar surface such as a column frit, column body or resin matrix. This binding is sometimes called nonspecific binding because although the false positive protein was recovered it was not due to the protein-protein interaction of interest. However, false positives can also be due to secondary interaction of the antigen or low selectivity interactions of the antibody. Secondary interactions are related to Co-IP complex formation. Low selectivity interactions are when an antibody can bind to several different proteins (albeit the protein of interest has the highest selectivity interaction).

There is one interesting note about false positives. They can be dealt with provided the results are consistent and predictable. In other words, if a material is recovered that is found to be nonspecific to the complex but can be recovered consistently and predictably, then the nonspecific material can be reliably dismissed as being not important. In many cases, false positives can be dismissed by identification and subsequent determination of their actual function. The protein may be identified by excising the band from the gel and analyzing by mass spectrometry.

What is a false negative?

A false negative is simply when the antigen is not recovered even though it is present in the sample. This can occur because the conditions for complex formation are not correct. However, this is more likely to happen because the antigen is low concentration and the recovered protein is therefore also very low concentration. Nevertheless, the tip concentrating effect of the PhyTip column can increase the concentration of the recovered protein by up to a factor of 10 over competing technologies.

How is immunoprecipitation (IP) related to co-immunoprecipitation (co-IP)?

Immunoprecipitation is closely related to co-immunoprecipitation. In co-IP, intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) are captured and purified by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. Targeting this known member makes it possible to pull the entire protein complex out of solution and subsequently, by using mass spectrometry, identify unknown members of the complex.

Co-IP works best when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple members of the complex out of solution. This process of pulling protein complexes out of solution is sometimes referred to as a “pull-down.” Identifying the members of protein complexes may require several rounds of precipitation with different antibodies for a number of reasons. A particular antibody often selects for a subpopulation of its target protein that has the epitope exposed, thus failing to identify any proteins in complexes that hide the epitope. In many cases, only less than half of the proteins of a given complex are captured with a single antibody. However, the first round of co-IP will often result in the identification of many new proteins that are putative members of the complex being studied. The researcher will then obtain antibodies that specifically target one of the newly identified proteins and perform a new co-IP experiment. Likewise, this second round of precipitation may result in the recovery of additional new members of a complex that were not identified in the previous experiment, and so on. As successive rounds of targeting and co-IP are performed, the number of identified proteins will continue to increase. The identified proteins may not ever exist in a single complex under a given set of conditions. Rather, they may represent a network of proteins interacting with one another at different times under different conditions for different reasons.

How is IP related to chromatin immunoprecipitation (ChIP)?

Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. This technique provides information regarding the protein–DNA interactions that occur inside the nuclei of living cells.
The in cellulo nature of this method is in contrast to other approaches traditionally employed to answer the same questions. By using an antibody that is specific to a putative DNA binding protein, one can immunoprecipitate the protein–DNA complex from cellular lysates. Crosslinking is often accomplished by applying formaldehyde to the cells (or tissue) or by using DTBP, for example. Following crosslinking, the cells are lysed and the DNA is sheared by sonication. Then an antibody is used in the sample in the same manner as in IP to capture a protein and its associated (crosslinked) DNA fragment. After reversing crosslinks, the DNA can be identified by sequencing. The location on DNA that the specific protein binds to can then be determined by
PCR, qPCR, sequencing, or microarray analysis