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Co-IP Immunoprecipitate - 1 Kit +1 Artifact Can Help You easily
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Co-ip immunoprecipitation is A classic method to study the interaction between proteins. Based on the specific action between antibody and antigen, Protein A/G in beads is conjugated to Protein A and antibody complex, and then B Protein potentially interacting with Protein A is also precipitated. Generally, WB analysis was performed on the obtained immune complexes.
 Immunoprecipitated co-IP schematic diagram
The operation process of co-IP itself is not complicated, but the trouble is that the relevant operation steps need to be optimized according to the actual experimental situation. After a while of hard work, the co-IP you get is likely to look something like this.
 Co-ip's high background and antibody contamination problems
The problems of high background and antibody contamination (IgG cross-reaction) of co-ip with co-immunoprecipitation are quite headache. The following are the optimization suggestions to you:
1) High background for nonspecific effects
There are a large number of proteins in cell lysates that may bind nonspecifically to target complexes during manipulation. These non-specific interactions are generally removed by thorough cleaning of the immune complexes in the Beads binding, but other strategies can be applied to optimize the non-specific binding, such as:
▲ Changing the ionic strength of the buffer by titrating the salt concentration from 120mM to 1000mM;
▲ Reduce the use of primary antibody until the signal interference ratio is maximized;
▲ Pre-clearance of cell lysates is performed according to our recommended procedures.
2) For antibody contamination (IgG cross reactivity)
The problem of co-immunoprecipitated co-IP antibody contamination is the interference of IP antibody IgG bands (occurred when IP antibody and WB antibody are homologous) in WB analysis. The light and light chains of IP antibody are around 25kD and 50kD, and there may be < in SDS-page. 5kD deviation. In this case, the following methods can be used to optimize:
▲ IP and WB use different sources of antibodies, IP is mouse source WB can choose rabbit antibody;
▲ The use of specific recognition of IgG light chain or heavy chain secondary antibodies, such as the target protein and IgG light link close, can be used heavy chain secondary antibodies;
▲ The IP antibody and Protein A/G-beads were cross-linked with cross-linking agent. The target Protein - antibody - Protein A/G-beads complex was treated by adding sample buffer without mercaptoethanol. Finally, the complex was removed by centrifugation, leaving only the target Protein in the supernatant;
▲ Before WB analysis, low pH elution can be considered instead of boiling. Low pH (2-3) can reduce the antigen/antibody interaction, so as to separate the antigen and antibody (e.g. 1M glycine buffer, pH 2-3), pay attention to pH balance before electrophoresis;
▲ Incorporation of commonly used fusion tags (such as GFP) into the main target proteins of co-IP, selection of high-quality specific anti-fusion tag antibodies, if necessary, can also be pre-fixed for protein complex purification.
Facing the high background of co-IP and antibody pollution, DIY is a kind of spirit, but we recommend 1 Kit +1 artifact to help you easily finish!
 abs955 Immunization (total) Precipitation (IP/CoIP) Kit
The bestsellingabs955 immunoprecipitation (IP/CoIP) kit，from ibsin uses high-quality Protein A/G agarose beads with high cost performance, well-validated protocol and supporting auxiliary reagents to facilitate your IP/CoIP experiment in A one-stop way!
In addition to this cost-effective co-IP pro kit, Chromotek's GFP-Trap ®!
 Schematic diagram of co-IP results after using GFP-Trap ®
GFP- Trap®is based on antibodies from the Camellia family (camel, dromedary camel, non-humpback camel and alpaca) that bind to antigens via the VHH domain and are chemically stable with high specificity and affinity against existing antigens. Gfp-trap ® is the ideal choice for the validation and isolation of GFP fusion proteins and their interaction proteins.
 Chromotek is an excellent choice for cell biology and proteomics
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March 15, 2022
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