ChromoBlog

How to prepare protein interaction partners for MS analysis

Posted by Christoph Eckert on Apr 24, 2017 11:02:03 AM

Immunoprecipitation (IP) followed by mass spectrometry (MS) analysis is a powerful method to identify interaction partners of a protein of interest. However, sometimes it can be difficult to obtain reliable results.

Now, the combined use of ChromoTek GFP-Trap for immunoprecipitation and PreOmics iST sample preparation kit is in fact a straight forward, reproducible, and reliable approach to identify protein interaction partners. It even preserves posttranslational modification (PTM) depending protein-protein interactions.

In a recent applications note we have demonstrated how protein interaction partners of PARP1 can be identified: We have immunoprecipitated eGFP-tagged PARP1 protein and its interacting partners using the GFP-Trap_M and subsequently processed the pulldown for MS analysis following the instructions of the iST kit.

PARylation mediates the interaction of PARP1 to several DNA repair proteins. Our results show that those interactions are not disrupted by neither the IP nor the MS sample preparation used herein. Therefore, the streamlined combination of the GFP- Trap and the iST kit’s workflow proves to preserve PTM-mediated protein-protein interactions.

 

Download application note

 

The PreOmics iST kit can also be used with other ChromoTek Nano-Traps.

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Topics: GFP-Trap, Immunoprecipitation, Mass spec, GFP Immunoprecipitation, GFP MS

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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