Immunoprecipitation / IP protocol

Immunoprecipitation / IP general procedure is use antibody to bind special protein, when the antibody was conjugated to a solid phase(magnetic beads, agarose,and so on). 

Target antigens are usually immunoprecipitated from complex solutions, such as cell lysates, the goal being to isolate and eventually detect and measure a specific protein (i.e., the antigen of the specific antibody). The basic protocol for performing an is diagrammed below, where the order of steps can be done in two different ways. 

In one sequence, an antibody (monoclonal or polyclonal) against a specific protein is pre-immobilized onto an insoluble support, such as agarose or magnetic beads, and then incubated with a cell lysate containing the target protein. During the incubation period, gentle agitation of the lysate allows the target antigen to bind to the immobilized antibody. The immobilized immune complexes are then collected from the lysate, eluted from the support and analyzed based on the nature of the target antigen.

Alternatively (right), free, nonbound antibody is allowed to form immune complexes in the lysate and then the complexes are retrieved by the beads. While the pre-immobilized antibody approach is more commonly used for, using free antibody to form immune complexes is beneficial if the target protein is present in low concentrations, the antibody has a weak binding affinity for the antigen or the binding kinetics of the antibody to the antigen are slow.

RNA-IP Complex IP Chromosome IP
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Immunoprecipitation / IP introduction
What is IP?
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What is ChIP?
What is RIP?
Immunoprecipitation / IP protocol
Classic IP Protocol
Complex IP
Chromatin IP
RNA IP Protocol
Magnetic beads IP Protocol
Immunoprecipitation / IP tips
Immunoprecipitation / IP troubleshooting
1. No Proteins Detected following IP
2. High Background (Specific or Nonspecific)
3. Antibody Fragments Blocking the Signal of Interest
4. The Worst Case Scenario: Co-IP is Not Successful
Immunoprecipitation / IP Antibody FAQ
Non-specific binding
Usage of IP antibody & Cell lysate
No detection of target protein
High background
Heavy chain & light chain
Choice of protein A/G/L
Protein A/G/L blocking
Are the sinobiologcal Immunomagnetic beads Protein A, Immunomagnetic beads Protein G and protein Lwere pre-blocked with BSA?
Extracellular protein IP antibody
Nuclear protein IP antibody
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High molecular IP
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Protein interaction & IP
ELISA & Immunoprecipitation / IP
Antibody purification & Immunoprecipitation / IP
Protein purification & Immunoprecipitation / IP
Chemiluminescence & immunoprecipitation / IP