ChromoBlog

Co-immunoprecipitation troubleshooting

Posted by Dr. Astrid Sitte on Aug 27, 2021 5:21:16 PM

Co-immunoprecipitation (Co-IP) describes the isolation of a protein and its binding partners from a cell extract using a Nanobody or antibody that is bound to beads. The protein, that directly interacts with the Nanobody, or antibody beads is called “bait”. The binding partner that is indirectly precipitated is called “prey”.

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Topics: GFP-Trap, Immunoprecipitation, Co-IP

How to conduct a Co-immunoprecipitation (Co-IP)?

Posted by Dr. Astrid Sitte on Jul 30, 2021 2:39:52 PM

Co-IP describes the immunoprecipitation of a protein of interest and its interacting partners.

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Topics: GFP-Trap, Immunoprecipitation, Co-IP

5 Tips for better immunoprecipitation (IP)

Posted by Christian Linke-Winnebeck on Jul 21, 2016 1:10:15 PM

Pull-down of proteins can be difficult, particularly when they are expressed at low levels. Here we present 5 tips, which will considerably improve your IP results.

 

  1. Protein concentration matters

The higher the protein concentration, the higher the IP yield. Try to use a concentration as high as possible.It makes a significant difference if the concentration of your protein during IP is 1 nM (low protein expression level), about 50 nM (intracellular endogenous protein expression level, will be diluted by buffer for IP) or 1000 nM (high protein concentration).

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Topics: Nano-Trap, Immunoprecipitation, Co-IP, GST pulldown, MBP pulldown

New Nano-Trap: Focus on MK2!

Posted by Kourosh Zolghadr on May 13, 2014 4:09:00 PM

MAP kinase-activated protein kinase 2 (MAPKAP-K2, MK2; Gene ID: 9261) is a 400 AA (46kDa) large enzyme that plays a central role mainly in the inflammatory response and cytokines production. It belongs to the serine/threonine-protein kinase family and is also involved in endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling, DNA damage response and transcriptional regulation.1-4

Following stress, it is phosphorylated (at Thr-222, Ser-272 and Thr-334) and activated by MAP kinase p38-alpha/MAPK14, leading to phosphorylation of substrates.5 Phosphorylation of Thr-334 (located between the kinase domain and the C-terminal regulatory domain) may serve as a switch for MK2 nuclear import and export. Phosphorylated MK2 masks the nuclear localization signal at its C-terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.6, 7

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Topics: Nano-Trap, MK2-Trap, Immunoprecipitation, Co-IP

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