Technique / Proteomics and protein biochemistry / Immunoprecipitation IP

Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity. Co-immunoprecipitation (also known as a pull-down) can identify interacting proteins or protein complexes present in cell extracts: by precipitating one protein believed to be in a complex, additional members of the complex are captured as well and can be identified. The protein complexes, once bound to the specific antibody, are removed from the bulk solution by capture with an antibody-binding protein attached to a solid support.

General Principles of Immunoprecipitation new recommended protocol
This is the Bart's cookbook on immunoprecipitaiton. It gives some general principles on performing immunoprecipitation using different lysis buffers.(Sefton Lab, Salk Institute). Content: RIPA buffer NP40 buffer PBS TN Boiling in SDS Antisera ...

Anti-FLAG Co-Immunoprecipitation protocol
Immunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has been raised. This primary antibody is either already bound to agarose or can be bound to the protein A/agarose beads during the procedure in order to ...

CO-IMMUNOPRECIPITATION ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS protocol
This protocol uses total cell extracts to analyze putative protein-protein interactions in eukaryotic cells. One can also use nuclear extracts as a source for this protocol (Morris lab at University of Pennsylvania). ...

Immunoprecipitation protocol from Sigma protocol
Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications: to determine the molecular weights of protein antigens, to study pro ...

Phosphatase inhibitors cocktails selection guide new review
Selection guide for phosphatase inhibitors cocktails (Merck). Phosphatase inhibitor cocktails is used to prevent dephosphorylation of proteins in protein biochemistry applications. Commonly used protein phosphatase inhibitors protect dephosphorylation of se ...

Protein A Sepharose Regeneration troubleshooting
I am using Protein A Sepharose (Pharmacia) for immune precipitations. I elute immune complexes after binding with laemmli buffer (SDS-PAGE loading buffer). Is there anyone who has experience, whether this very expensive material can be regenerated ...

How to remove RNase from immunoprecipitation? troubleshooting
We are trying to do a "simple" immunoprecipitation in which the unbound fraction will be assayed for RNA processing activity. We find that protein A-agarose beads have little non-specific RNase activity. After adding serum (immune or non-immune), we w ...

Immuno-precipitation of Phospho-Tyr proteins troubleshooting
I am trying to assess the degree of phosphorylation a receptor undergoes in response to it's ligand plus various other factors. If I apply the ligand to my cells and then extract in the presence of sodium orthovanadate and do a western using an antibo ...

Immunoprecipitation western blot troubleshooting troubleshooting
In an immunoprecipitation-western blot I used rabbit polyclonal antibody and protein A agarose beads(protein A is covalently bind to beads) to pull down antigen complex. In following western blot I used rabbit polyclonal antibody as primary antibody and HRP-an ...

troubleshooting tips of IP, western and immunostaining hot troubleshooting
General troubleshooting tips of immunoprecipitation, western blot and immunohistochemistry from Chemicon. Link: troubleshooting tips of IP, western and immunostaining This link was lasted ...

Troubleshooting Tips for Immunoprecipitation / Kinase Assays new troubleshooting
A brief troubleshooting guide on immunoprecipitation - kinase assay. (Upstate) Determine the optimal stimulation time and temperature for each individual kinase that is to be tested. This is essential since optimal stimulation periods are often short and ca ...

Immunoprecipitation TroubleShooting Guide new troubleshooting
Troubleshooting guide from Sigma. It addresses immunoprecipitation problems on: 1. No binding 2. High background or unwanted precipitates ...

co-immunoprecipitation question: starting concentration troubleshooting
What is a good starting concentration of each protein to have? What volume? I know that my proteins are tight-binding, so dissociation should not be a big problem. (post from NWFSC forum). ...

Immunoprecipitation troubleshooting guide troubleshooting
A Immunoprecipitation troubleshooting guide from Stressgen Biotechnologies. ...

Troubleshooting the immunoprecipitation procedure new troubleshooting
A troubleshooting guide on Immunoaffinity purification (Immunoprecipitation) procedure from Roche. ...

Immunoprecipitation troubleshooting new troubleshooting
A troubleshooting guide on immunoprecipitation. (Agrisera) Problems: No binding High background signal ...

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