Co-immunoprecipitation

1. Overview

Methods like immunoprecipitation(IP), co-immunoprecipitations (Co-IP)are important in relation to study a specific protein of interest and their interaction partners in cells or tissue sample. These methods also give insight about the upregulation or downrefulation status of the proteins, posttranslational modifications of proteins or their molecular weight. In immunoprecipitation a particular antigen component of protein is isolated from thousands of proteins with the help of antibody which can specifically binds to the antigen of interest. Immunoprecipitation thus uses antibody which is attached with magnetic beads or agarose-resin beads to isolate antigen and subsequently detected by methods like western blotting. Immunoprecipitation generally works for an individual protein whereas, when an antigen is present in a complex association with other protein or ligands, they are immunoprecipitated by Co-immunoprecipitation or Co-IP. Both IP and Co-IP are based on functional similarities except the fact that IP is applied for individual protein isolation whereas, immunoprecipitation of intact protein complexes is referred as co-immunoprecipitation.  Immunoprecipitation method is generally applied for isolating known proteins but, Co-immunoprecipitation methods isolates unknown proteins which are present in complex formation with known protein. Thus, Co-immunoprecipitation works effectively when the proteins involved in the isolation have the ability to tightly bind with one another. This helps in pulling out multiple members of the complex by latching onto one member by using an antibody. Usually, this is orchestrated when one protein in the complex is known and a suitable antibody is used. By targeting the known antibody, it becomes easier to pull the entire protein complex from the solution. For the same reason, the technique is sometimes referred as pull-down immunoprecipitation. The antibody which is known is referred as Bait protein and the protein complex to be identified is termed as prey protein. The technique is primarily used by molecular biologists to study protein-protein interactions. With the help of the Bait protein and the antibody which interacts with it, Co-IP methods become the method of choice for identification of novel protein.protein interaction and their validation studies. Specifically, in situations where cells get lysed woth denaturing reagents they are able to retain the inracellular protein-protein interactions. Co-IP methods are thus physiologically relevant methods for identification of protein.-protein interactions by an indirect approach.

2. Principle

As Co-Immunoprecipitation originates from immunoprecipitaiton techniques, it shares the same principle of antigen-antibody specificity/reaction as in any Immunoprecipitation technique.  When a cell is lysed in non-denaturing condition, it retains the proteins in their native complex. Thus, according to the image the known protein in the complex X is referred as bait protein and the interacting protein Y is known as prey protein respectively. In a normal cellular environment, protein X will get attached to protein Y and the complex protein-protein Y is precipitated or isolated in presence of an antibody which can bind specifically to protein X. At the end, Co-IP thus gives information whether protein X and Y are interacting with each other or Y as a new interacting partner of X and also provide supports related to the information. The method starts with the lysis of cells or tissues which are known to have the protein or interacting partners of interest .Lysis buffers which contains non.ionic detergents are thus preferred as to retain the native ineractions status. The Bait protein is isolated from the lysed sample and immunecomplex containg prey proteins thus get captured with the bait protein. The immunecomplex containing a mixture of Bait protein, Bait-Prey protein and antibody is gets precipitated while using beads such as sepharose, agarose or magnet. A washing step next frees the resulting immunocomplex from irrelevant binding proteins, non-binding proteins. Further the precipitated partners are identified by methods like SDS-PAGE separation or mass spectrometry. In principal, Co-IP can be demonstrated as an extension on IP methods for isolating and purifying targets which identify secondary targets like interacting proteins in addition to primary antigen.

3. Applications

Immunoprecipitation method was originally invented as an alternate to affinity chromatography for small scale protein production. This method is a popular choice for identifying and separating protein with low abundance. Co-immunoprecipitation is a well known and popular method for studying protein-protein ineraction invitro. The main applications are

  • Prove interaction between two proteins
  •  Identifies a new protein by using a known one.

4. Advantages

  • In Co-IP method proteins which are interacting are generally Post-translationally modified and conformationally natural.
  • Proteins interact in almost physiological condition.
  • This method is highly specific in nature and at the same time quite simple to perform.
  • The eluted protein complex is compatible with all applications which are used downstream such as western blot or mass spectrometry.
  • Reagents which are used for Co-IP can be used several time thus making it cost effective.

5. Disadvantages

  • Weaker signals from low affinity proteins are not detected.
  • Not suitable for identification of protein interaction which takes place within short time period.
  • Antibody selection is critical and target protein prediction needs to be correct
  •  There could be a chance that two proteins are not directly interacting each other. There might ne presence of  a third protein and the presence of  such  third protein in certain protein-protein interaction could weaken the procedure
  • Antibodies with high affinity or avidity are often difficult to isolate.
  • The interacting protein identification is mostly based on the prediction of interaction between Bait and Prey antibody. This a wrong interpretation of Bait antibody will results no interacting partner while performing the method.

6. Protocol

  • 15ml centrifuge tube
  • Microcenrifuge
  • Rotating shakers
  • Protein A/G beads
  • Equipments

7. Workflow of Co-Immunoprecipitation

 The steps of Co-immonuprecipitation goes through several steps such as cell lysis, pre cleaning of beads, binding of antibody to immune complex, washing, elution of target protein and detection of immune complex.

Cell lysis: Cell lysis is the first step in the Co-immunoprecipitation method. The choice of buffer should be mild as they should not interfere with the protein-protein interaction, but at the same time required to be harsh so the buffer should be able to extract out the protein.

Pre-cleaning: Pre-cleaning procedure is important for reducing background generated by immune complex formation. Without any valid reason, pre-cleaning step should not be omitted.

Binding of immune complex:  Binding includes complex formation between antigen, antibody and beads. Buffers which is used in immunecomplex formation and washing are the critical factor for binding process. The order, how the components are attaching to each other is a crucial factor for successful Co-IP. One way is to add the antibody to the beads and next add the lysate to the antibody-bead complex. Otherwise, antibody is first incubated with the cell lysate to form antibody-antigen complex and next beads are added to pull down the immune complex.

Washing: Washing is an important step, as this will wash of the non-specifically binding protein retaining the specific immune partners. If background problem persists while detecting, a more stringent wash buffer will help to obtain purified complex.

Elution of protein complex:  For elution harsh environment such as loading buffer with SDS, or mild elution buffer like Glycine buffer is used.

Detection: Interacting protein partners are detected by methods like Western blot or Mass spectrometry.  Western blot method is based on the detection of desired protein with the help of detection antibody. As the amount of precipitated protein is not too high, a sensitive substrate is required for western blot technique.

On the other hand immunoprecipitation in a combination of mass sprectrometry or IP-MS is a direct method of identifying the target protein with the help of peptide sequence.

Controls in Co-immunoprecipitation:  For a successful Co-immunoprecipitation experiment, incorporation of positive and negative controls is necessary. Total cell lysate can be used as positive control. Alternatively and protein which is not related to target protein can also use as negative control.  Positive control confirms whether the assay was successful or not. Whereas, empty beads combined with cell lysate without the antibody can be used as negative control. When there is no bound IgG is present with the lysate-bead complex, it should not generate any bands in detection method like western blot. Alternatively antibody used in immune complex capturing can be added to the lysate without the beads, as this will indicate antibody cross reactivity. Another approach is to bind empty beads with lysate to check if there is any interference is created from the beads.

8. Detailed procedure

The process starts with the transfection of cells with the protein expressing plasmid for the production of Bait protein or protein of interest. Transfection protocol is required to follow as per the instruction of transfection agents.

  1. Generally after 48 hours the transfected cultured adherent cells are carefully washed two times with chilled PBS twice. Non adherent cells are washed with PBS and centrifuge at the rate of maximum of 800-1000 rpm for five minutes for pelleting the cells.
  2. Next, ice cold RIPA Lysis buffer with protease inhibitor cocktail is added to the culture plate (1ml for 107 cells).
  3. The cells are scrapped off from the culture plate to a5ml precooled Eppendorf tubes with a clean and ice cold scraper.
  4.  After this step, the cell suspension is incubated in low-speed rotating shaker for 15 mins at 4°C to lyse the cells.
  5. Next, the cell suspension is centrifuged in the eppendorf at 14,000 g 4°C for 15min, the supernatant is transferred to new tubes immediately and placed on ice. The pellet is discarded. The supernatant can be stored as -80oC for a longer time period.

The critical parameter for successful Co-IP is to maintain the protein-protein interaction in their native physiological state. Thus all work with cell lysis buffer should be performed at low temperature, and in the presence of non-ionic detergents in lysis buffer. During a Co-IP assay, location of target protein should also be considered. For instance, target proteins which are located inside the organelle are required to release before the protein is interacting with the antibody. On the other hand, if the target protein is located on the membrane, their extraction will depend on the detergent type and concentration. Types of detergent in lysis buffer are also dependent of property of target protein.

Centrifugation force applied to make the cell supernatant  may require to adjust based on cell type.

  1. In the meantime, agarose beads are prepared for the next step. Generally, the agarose A/G beads are resin beads which are used for purification of antibody these beads are consisting of recombinant fusion protein A/G which has been immobilized on 6% agarose and are suitable for purification of IgG immunoglobulins. Usually the protein A/G-agarose beads are washed twice with PBS and a 50% protein slurry of A/G agarose working solution (in PBS) is made. It is advisable to cut the front of the tip, so thus beads will not be disrupted.
  2. Next, 50% protein slurry of A/G agarose is added with ratio of 100μl for a 1ml sample solution (supernatant).
  3. After this the eppendorf tube containing supernatant with A/G agarose bead is set to incubate in horizontal shaker for 10min at 4°C. This step is done mainly to eliminate non-specific binding proteins.
  4. Again, the tube is Centrifuged 14,000g at 4°C for 15min.
  5. At this stage, the supernatant is transferred to new centrifuge tubes and discard protein A/G-agraose beads.

Beads at this stage can be washed with lysis buffer to increase the amount of protein complex.  .

  1. The protein is next quantified by BCA assay or other protein quantification

A SDS gel can be run at this stage. Coomasie staining of the gel will show whether serum Ig has been removed properly from the bead-antigen complex. Presence of bands at the molecular weight of 25 and 50Kd indicates presence of antibody heavy and light chain which may hinder the immunoprecipitaiton in the next steps

  1. The protein is further diluted to 1μg/μl with PBS to decrease the concentration of detergents.
  2. The diluted primary antibody is added at an appropriate amount to 500μl total volume of the solution of supernatant-bead complex.
  3. Next, the antigen-antibody complex in centrifuge tube is incubated for overnight at 4°C in a rotating shaker.

For downstream enzyme assay after immunoprecipitaiton, a shorter incubation time is recommended.

  1. Centrifuge the tube at14,000g for 5s, keep the pellet and wash with pre-chilled washing buffer for 3 times. (800μl each)
  2. The supernatant is collected and analysis is carried out via SDS-PAGE, western-blot, or mass spectra analysis. Alternatvely the supernatant can also be stored at 4oC for future application.

Note: This protocol gives lower yield, but avoids the problem of co-elution of antibodies. If higher yield is expected, the antibody can be mixed with protein sample prior to addition of Protein A/G-agarose beads. The antibodies can co-elude with target protein and interference can be observed in western blot detection.

9. Preparation of Reagents and Buffers

  1. Phosphate Buffered saline,1X PBS
  2. RIPA lysis  buffer is made of Tris-HCl: 50 mM, pH 7.4, Nonidet P-40 (NP-40): 1%, Deoxycholate Na:25%,, NaCl: 120 mM, EDTA: 1 mM, PMSF: 1 mM, Leupeptin 1 μg/ml, Aprotinin 1 μg/ml, Pepstatin1 μg/ml, Na3VO4: 1 mM, and NaF: 1 mM. ( PMSF, leupeptine, Aprtonin, pepstatin should be added freshly to the lysus buffer  Amount the NaCl should no exceed 1M:
  3. Washing Buffer made of tris buffer saline with tween 20.
  4. protein A/G-agarose beads
  5. Specific antibody (MAb or PAb)
  6. Elution buffer: SDS-PAGE sample loading buffer or Glycine buffer.

10. How to Optimize Co-Immunoprecipitaiton?

Although the principal of Co-IP is easy to perform, however, there are few circumstances which may interfere with the protein-protein interaction in physiologically relevant conditions.

Binding: As in Co-IP cells go through mechanical and chemical procedures, incubations and washing steps, it is very necessary that the protein-protein interactions maintain stability. Thus in situations where the protein-protein interaction is transient or affinity in low, Co-IP might not perform able to detect the interaction partners. Thus an important step for retaining the interaction is cell lysis and washing. While choosing the buffer it is important to choose low ionic strength buffer with detergents which are non-ionic in nature like NP-40 so the target protein complex remain unaffected. In addition, gentle handling is also required during centrifugation and washing steps to prevent loss of protein complex. During the assay period, it is essential to keep the lysate or supernatant on ice and addition of protease and phosphatase inhibitors are highly recommended to as there are chance of proteolysis or dephosphorylatiion of proteins exists. Repeating freezw thaw cycle can damage the protein-protein interaction, thus working with fresh lysates are the best option for desired result production.

Nonspecific binding: As in the cells there are lot of proteins it is inevitable that nonspecific binding will takes place. In addition to this, during the lysis steps cellular compartmentalization of a cell get destroyed, there is a high chance that proteins like actin which is highly abundant might bind and interfere with target protein. Washing is thus most important criteria to get rid from the nonspecific interactions. However, other options such as using higher ionic strength lysis buffer or lower amount of primary anibody or a precleaning step of cell lysate may reduce the risk of nonspecific binding.

Selection of antibody: As antibody is the important part of the immunoprecipitation assay, is it always advisable to use antibody of different origin for the capturing and detecting methods, such as anti-rabbit antibody for the pulling down of protein complex and anti-mouse antibodies for downstream detection like western blots. This will help to reduce the chance of interference generated from heavy and light chain of the antibody which is used to isolate the protein complex. Apart from use of antibodies from different species, polyclonal antibodies are also preferred for a higher binding of antibody to protein complex. In comparison to monoclonal antibodies, polyclonal antibodies have the capacity to get attached with the multiple epitopes expressed in target proteins. This will lowered the chance of protein complex being washed away during the washing steps. Whereas during detection, monoclonal antibody is preferred for specific detection.

Contamination of antibody: Antibody contamination is a common problem in Co-immunoprecipitation. Downstream applications such as Western Blot may generate additional bands in the gel. The light and heavy chain antibodies which might have co-eluted with the protein complex and interfere with the identification process. One way to get rid of the problem is covalent binding of antibody with the beads. This method also allows the reuse of beads coated with antibody. Another strategy used to reduce cross contamination of antibody is use of biotin-streptavidin system, where biotinylated antibody is used for target protein whereas streptavidin is used to coat the beads.

Choice of beads: There are few types of beads available for the use in immunoprecipitation methods. Among them protein A and protein G conjugated beads are widely used. Both protein A and G has high affinity towards antibody, but depending on the cell type and target organisms, they will show difference in antibody binding. In general protein A beads are working better with rabbit antibodies, while protein G for mouse antibodies. Apart from protein A/G beads, agarose and magnetic beads are commonly used in Co-IP. Agarose beads have a porous sponge like structure and they shows high affinity towards antibody. The use of agarose beads is also cost effective. However, as antibodies get highly trapped in this porous structure and become difficult to wash out. Thus while using agarose beads extensive cell lysate cleaning and washing is required for background reduction. Additionally agarose beads are also required longer incubation period as porous structures results in lower diffusion rate. On the other hand, magnetic beads are small with smoother outside. As a result they have low binding capacity and required a large volume of beads. But they do not require any centrifugation and less washing steps. Magnetic beads showed increased diffusion rate and results in lower incubation time with antibody.  Altogether they are a good option for used in Co-immunoprecipitation.

11. Frequently Asked Questions

1. What are the different types of immunoprecipitation?

  • Individual protein immunoprecipitation

This method involves separation of a single protein from several other proteins present in a solution. This sample ranges from crude cell extract of animal and plant, body fluids. In individual protein immunoprecipitation, the target protein is known and antibody specific for that protein is used to isolate it.

  • protein complex immunoprecipitation

This method involves immunoprecipitation of a complex containing antigen and related protein or ligand which remains bounded with it. Protein complex immunoprecipitation is also known as Co-Immunoprecipitation, When several proteins are present is n complex and one of its members is known, then unknown proteins can be identified on the basis of known member with the help of an antibody which is able to bind with the known member.When the members of the protein complex are interconnected with each other then it is easier to isolate the whole complex together and identify the unknown member. This method sometimes also called as pull down.

  • chromatin immunoprecipitation

Chromatin precipitation or CHIP is method to identify protein and DNA interaction within cell. This method is utilized to known which genomic regions are associated to a particular protein or whether several proteins are associated with a particular genomic region.CHIP assay is also useful for the identification of recruitment order of protein in the promoter region or histone modification for a particular genomic region Additionally CHIP also give information related to binding of transcription factor.

  1. RNP Immunoprecipitation

Gene expression which is regulated by ribonucleoprotein complexes is post-transcriptionally regulated. Ribonucleoprotein complex includes RNA binding proteins and non coding RNAs and together they regulate cell functions such as splicing, stability of mRNA, nuclear export or protein localization.  Likewise mRNA which is functioning on protein synthesis for specific cellular process are found to associated with ribonucleopeotein complex forming mRNP. These RNA binding proteins which is essential for RNA functions are also important as a target of potential therapeutic options. RNP imunoprecipitation or RNP-IP is thus works to isolate the miRNA, mRNA from the ribonucleoprotein complex with the help of the antibody targeted against RNA binding proteins.

2. What are the different methods of immunoprecipitation?

Immunoprecipitation is performed by both direct and indirect methods.

Direct method:  In this method the beads are first bound to antibodies and next the antibody-bead complex is added to the protein mixture and incubated with a rotation with a temperature in-between 2–8°C. The time of incubation is entirely dependent on the target protein concentration and how specific the antibody is behaving towards the target protein. Next the target proteins are captured by the beads through antibodies, or simply immunoprecipitated. The antibodies are immobilized on solid phase substrate such as microscopic agarose and superparamagnetic microbeads.

The direct method is suitable only if the target protein is present in higher amount or there is less requirement of antibody.

Indirect method:  Here first the antibodies are added to the cell lysate, so a protein complex is formed and antibody is bind to target proteins. Beads coated with protein A/G is added subsequently to target bound proteins in the complex. Beyond this point, the protocols converge and as the end-results are one and the same. However, excess amount of antibody can results in binding with A/G beads as free antibodies are moved faster than bound one. The indirect method is very applicable when the target protein is present at low concentration or the antibody has poor affinity for the target protein. To control the background binding, sample and beads are required to incubate for smaller time period.

3. What is chromatin immunoprecipitation?

Chromatin immunoprecipitation is used to study the relationship between proteins and DNA. The technique combines DNA sequencing techniques with immunoprecipitation to identify the binding sites of DNA-associated proteins. During CHIP assay cells are treated with formaldehyde, a cross linking agent which fixes the region of DNA.protein interaction. Next, cells get lysed and chromatins are broken down to small fragments with the help of sonicator or by enzyme digestion method. Immunoprecipitation is next followed with antibody specific to target protein or a modified histone. One the immunoprecipitation is complete, the cross-linked compound   gets reversed and purified DNA is obtained. Several downstream processes are next followed to identify the genomic region where the protein interacts. Relative abundance of genomic region where the target protein binds routinely identified by standardized PCR methods. Real time quantitative PCR is also often applied to measure quantitative measurement of enriched sites accurately. In lot of instances, CHIP is couple with microarray known as CHIP on chip method for genome wide analysis of DNA.proein interaction.

4. What is RNP Immunoprecipitation?

The prinicipal of RNP immunoprecipitation is quite similar to CHIP. RNP immunoprecipitation targets ribonucleoproteins; where a specific antibody is targeted against the protein of interest and by doing so pulled down and isolate the RNA bindinig protein as well as related RNAs. To study the physical interaction between RNA and ribonucleoprotein, at first cells are lysed. The target protein and associated RNA are next immunoprecipitated using antibody to target protein of interest. RNA-protein complexes are separated and in the identity of RNA is determined using cDNA sequencing. The native method of RNP-IP helps to identify the RNA and at the same time also state how abundant they are.

5. What is visible imunoprecipitation?

 Visible immunoprecipitaiton method is based on the principle of Co-immunoprecipitation, only the difference being the visual detection of the protein-protein interaction. Protein of interests are tagged either with GFP or RFP protein and immunoprecipitated with beads containing GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads instead of antibody. Now, when the beads are looked under microscope If there is protein-protein interaction they will be visualized by expression of GFP or RFP.

6. What is yeast two-hybrid (Y2H) assay?

In yeast two-hybrid (Y2H) assay uses two protein, one get bound to the DNA binding domain or DBD and the second protein is bound to activation domain or AD also known as Pry protein. If the proteins are interacting with each other, then a DBD and AD domain interacted and a transcription factor placed upstream of a reporter gene gets active. This approach is a cost saving direct protein interaction methods. In some instances, the Bait protein is useful for the screening of new protein interaction partner from a protein fragment library.

7. What are tagged proteins?

One inherent disadvantage in immunoprecipitation is the difficulty in generating antibody which will work for a single known protein. To counter this disadvantage, research groups often design tags focused on C or N terminal end of the target protein. Tags are in principal peptide sequence which is incorporated into a protein. Examples of tags include green fluorescent protein, Glutathione S transferase and the Flag tag.  Tags serve various purposes for attached protein.Affinity tag such as glutathione-S-transferase (GST) or poly (His) tags are used to purify protein from cell lysate with the help of affinity techniques. On the other hand solubilization tags like thioredoxin helps protein folding and inhibit precipitation of recombinant protein. Chromatographic tags like Flag-tag can be added to recombinant protein  where target protein lacks specific antibody against it. Additionally, visualization tags like GFP helps to make a visual representation of the target protein.

8. How MS-IP works?

MS-IP or mass spectrometry coupled with Co-immunoprecipitation is a direct approach for identifying the interaction partner isolated in Co-IP. For this, eluted sample from Co-IP is analysed by mass sprectrometry and the generated MS data is  analysed with bioinformatics software for the identification of the proteins. In addition to identification, IP-MS also helpful to produce fold enrichment of target protein between control and treated samples as well as with proteins which are off-target. For a successful IP-MS analysis, two very different antibodies are used. One antibody is specific to the target protein of interest, whereas the second protein is generally used to detect a well-known abundant protein which is not related to target and serves as the control of the experiment. Next, proteins which are detected by mass spectrometry method is plotted in a graph in respect two those two IP protein. In this analysis, proteins are divided into three groups;

  1. Background proteins- protein which is bound to specific antibody or beads appeared in this part. They usually stay in the middle of the plot.
  2. Negative control proteins- proteins that are interacting with negative control antibody are plotted along the x-axis of the graph and they do not react with antibody against the target proteins.
  • Positive control proteins- proteins which are specific to target antibody are plotted along the y-axis and these are the interacting partners of the target protein precipitated in the assay.

Once the data generated the interacting proteins they are further analyzed for enrichment study. However, there is some limitation of IP-MS method. In general some antibody which is acting to capture the target protein fails to bind the protein in their native confirmation. Although other detection techniques are able to identify the target protein in this situation, however MS-IP cannot identify the proteins in this situation.

9. How GST-pull down is different from Co-immunoprecipitation

 Co-immunoprecipitation works to immunoprecipitate protein in their native state and the proteins which remain in complex represents their natural state. However, Co-IP can not rule out whether proteins which are present in a complex are directly interacting with each other or through a third protein. Wheras, in GST pull down assay is a method of examining protein-protein interaction invitro. In GST-pull down assay,  a glutathione-S-transferase tag is added to the N.terminus of the target gene which will produce the desired protein. Next, this protein is pulled down by glutathione beads as they provide high affinity to GST. If the protein of interest are present in a complex of other proteins are also pulled out with the target protein.

12. Troubleshoot for Co-Immunoprecipitation

12.1. Background is high

12.1.1 During cell Lysis:   

  • Protein aggregation could cause high background. Use of freshly prepared cell lysate is recommended. When frozen cells are used, cells which are stored at -80oC are desired.
  • Do not leave the supernatant for longer time; it will develop insoluble proteins in pellet. Take out the supernatant immediately. When the solution is not clear, centrifugation will resolve the issue.
  • Fresh addition of protease inhibitor in lysis buffer is recommended.
  • Preclearance of lysate or pre-incubation of lysate with the beads will reduce the nonspecific binding.( reference-protocol
  • Extracting desired target protein from the cellular compartment where they are situated, rather than the whole cell will reduce the unwanted cellular debris in the lysate.

12.1.2 During washing:

  • Take care at the washing steps. Thorough washing at required step will help to rescue the background. Minimum of 3 washing steps with each of 5-10 minutes will reduce the nonspecific binding.
  • Take care of washing the beads several times before incubation with cell lysate.
  • Use detergents such as Tween 20 and Triton X 100 in wash buffer in a concentration range of 0.01-0.1%.
  • If the removal of wash buffer is not sufficient, remaining wash buffer in the centrifuge tube could results in unnecessary proteins.

12.1.3 During binding:

  • Use of antibody which has been purified with affinity grade purification method.
  • Check the antibody concentration. Use of less amount or diluted antibody.
  • Use of less amount of protein or cell lysate. Generally 10-500 µg of cell lysate give required results.
  • Preclearance of lysate or pre-incubation of lysate with the beads will reduce the nonspecific binding.( reference-protocol)

12.2. Antibody elution is high

When large amount of antibody is eluted with protein of interest, beads crosslinked with antibody prior iunoprecipitation will reduce the eluted antibody amount.

12.3. There is no or less amount of target protein

  • Look for the desired protein expression profile in target cell or tissue. When protein expression is low, higher amount of cell lysate will increase the chance of protein product.
  • Look for recommended amount of antibody in datasheet, as lower amount than recommended will result in no capture of protein complex.
  • Check whether protein has been eluted from the beads. Use of specified elution buffer is recommended.
  • Bead and antibody isotype is matched.
  • Use of proper lysis buffer to lyse the cell.