The Protein Man's Blog | A Discussion of Protein Research

Following up the Immunoprecipitation Protocol: The Basics, we explore the IP protocol from the viewpoint of some of the protocols critical parameters.

1. Antigen extraction: As mentioned in the general protocol, cellular antigens are required to be extracted from a suitable source depending on the experimental requirement. The extracting method should be able to make antigens accessible for binding with the specific antibody. The antigens should be in a physical form in order to allow its separation from other cellular components. Hence, extraction with nondenaturing detergents such as Triton X-100, Nonidet P-40, CHAPS, digitonin, or octyl glucoside can allow the exposure of epitopes in the antigen for better binding with antibodies. Some of these detergents can even preserve weak protein-protein interactions, for example, digitonin. Some antigens are insoluble in non-denaturing detergents such as cytoskeletal structures, chromatin, membrane “rafts” or the epitope is hidden between the folded motifs of the proteins, and in that condition it is preferred to extract the antigens under denaturing conditions.
2. Antigen Concentration: The cellular abundance of the immunoprecipitated antigen is essential to determine efficiency of immunoprecipitation. Radio labelling or non-radiolabelled methods are suitable to detect antigens as low as 10,000 to 100,000 copies per cell, which is generally the case with signal transduction proteins, transcription factors or endogenous integral membrane proteins.
3. Pre-Clearing of the protein sample: In order to start immunoprecipitation, pre-clearing of the antigen sample is an additional step. The samples are incubated with the beads (Protein A and G or A/G) in order to get rid of unwanted antigens. The non-specific antigens get bound with the solid phase and hence can be removed by low-speed ultracentrifugation.
4. Antibody purification method: Polyclonal antibodies used for IP could be in the form of whole serum, ammonium sulphate–precipitated immunoglobulin fractions, or affinity-purified immunoglobulins. Among all these, affinity-purified polyclonal antibodies are preferred as they are relatively specific and give lesser background.
5. Antibody selection- Polyclonal versus monoclonal: The selection of type of antibody most suitable for IP is most important, though it is not an easy way.

Type of Antibody	Pros	Cons
Polyclonal antibodies	can frequently recognize multiple epitopes of the targeted antigen, hence gives a large spectrum of identification. enhanced avidity of the antigen-antibody interactions. minimal denaturing extraction is required as the chances of epitope exposure are higher.	polyvalent antibodies have the probability of cross-reactivity. Hence, it may lead to misidentification and higher background. requires highly efficient and expensive methods to purify.
Monoclonal antibodies	antigens do not require to be purified to serve as immunogens. less likely to cross-react with other antigens. requires less titre of antibody. produces minimal background.	recognises a very specific epitope hence require denaturing conditions sometimes to access the epitope. affinity of the antibodies get compromised sometimes during the production of the antibody, hence low affinity monoclonal are not suitable for IP.

Hence, an informed empirical approach should be used to choose the antibody to get satisfied results.  

6. Antibody titer: The titration of the antibody is required to get an optimal result. Too less of antibody will give inefficient immunoprecipitation while too much of antibody can create nonspecific IP because of increased amount of immunoglobulins bound with the beads.
7. Immunoadsorbents: Immobilized proteins (protein A, G, A/G and recombinant proteins) are efficient for antibody adsorbent and provide a beaded support. Click here for a comparison of these immobilized proteins in terms of Immunoglobulin binding.
   Both Protein A and protein G are relatively stable and can be sedimented easily by low -speed centrifugation. Although, some polyclonal or monoclonal antibodies may not efficiently bind with all protein A or protein G, which can be solved by using an intermediate antibody to the immunoglobulin of interest.
8. Nonspecific controls: For correct interpretation of the result, it is critical to include appropriate controls for any experiment. For Immunoprecipitation, appropriate nonspecific controls with the specific samples are used. Generally two types of controls are used:
   1. Incubation of an irrelevant antibody similar to the experimental antibody which share biochemical similarity and belong to same species and immunoglobulin subclass. "No antibody" controls are strongly not advised.
   2. Perform an Immunoprecipitation from the cells/tissues that do not express a specific antigen along with an immunoprecipitation of the antigen-expressing samples.
9. Washing: The washing steps must be optimized for sufficient removal of unbound proteins. Additional washes are most likely to reduce antigen-antibody association while insufficient washes may lead to background. The last PBS wash is required to remove detergent from the complex which might reduce the resolution on SDS-PAGE and other components of the buffer.
10. Analysis of Immunoprecipitation result: The immunoprecipitated antigens can be analysed by different ways such as Western blotting, ELISA and mass spectrometry.

 Related Blog Posts

• Immunoprecipitation Protcol: The Basics
• Magnetic Beads for Immunoprecipitation
• Protein Immunoprecipitations: Which method to use?

The cardinal principle behind immunoprecipitation (IP) is the isolation of an antigen by interaction between the antigen and a specific antibody conjugated to a sedimentable matrix. Antigen source may vary from cells or tissues without labeling, radio labeled or tagged cells, labeled or unlabeled proteins isolated from subcellular fractionation or in-vitro translated proteins. Immunoprecipitation can also be helpful in protein fraction analysis separated by gel filtration or differential sedimentation based on density gradients.   In order to capture interacting antigens, antibodies developed from various animal species that could be either polyclonal or monoclonal, are used for immunoprecipitation. These antibodies are tagged  non-covalently with specific immunoadsorbents such as protein A–or protein G–agarose or covalently linked with a solid-phase matrix.

Protein Storage and Stability

What are Proteins?

As one of the multifarious macromolecules, proteins are complex and crucial for cellular functions. Proteins are polymers built of monomer subunits called amino acids connected by a specific type of covalent bond known as a peptide bond. The properties of the protein depend on the type of amino acids present in them. Although the primary structure of the protein comprises of the amino acid sequence, the functional properties of the protein crucially rely on the three-dimensional or tertiary structure. Similarly, protein modifications for e.g., glycosylation, phosphorylation, may change the properties and function of the protein. These modifications alter the local conformation and mediate folding or stability as well as drive proteins to different cellular compartments. Proteins also display remarkable variability in terms of structure and flexibility depending upon their folding patterns. Some proteins are relatively rigid hence can function as structural meshes or connective lines. Proteins with reversible conformational changes (polymerization or depolymerization) are crucial for protein-protein interaction, growth and the transmission of information from cell to cell or within the cell.

Affinity chomatography is a routinely used technique for purifying or enriching a protein or molecule of interest through its specific binding affinity. The target protein adheres to a particular ligand that has been immobilized on a solid support (usually beaded agarose resin). This process produces high selectivity, resolution, and capacity for the protein of interest. Traditionally, affinity chromatography is performed in column format. Where the sample is applied and eluted by gravity flow through a packed resin bed of one to several milliliters. However, gravity flow columns are time consuming and require constant attention while supernatant filters through the column and resin. This process can be accelerated with G-Biosciences’ FastPure™ Spin Columns (Mini and Midi) specifically designed for simple and efficient small scale protein purifications. FastPure™ Spin Columns combine the effectiveness of gel filtration, and the speed of centrifugation for quick and reliable protein purification.