ChromoBlog

Tips and tricks for immunoprecipitation of low abundant proteins

Posted by Dr. Astrid Sitte on Apr 14, 2021 4:03:04 PM

Protein abundance is crucial for immunoprecipitation

During an immunoprecipitation (IP), the protein of interest (POI) is pulled down with a specific antibody or Nanobody conjugated to beads such as agarose or magnetic agarose. Unbound sample content, for example, other proteins, cell debris, and lipids, is removed by washing, while the precipitated protein is enriched on the beads.

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Topics: GFP-Trap, GFP Immunoprecipitation

Which beads should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Sep 16, 2020 9:42:57 AM

ChromoTek's Nano-Traps are optimized for the immunoprecipitation (IP) of proteins and their interacting factors. Nano-Traps comprise of a Nanobody/ VHH conjugated to beads. For 3 Nano-Traps, e.g. GFP-Trap®RFP-Trap®, and Spot-Trap®, we offer different types of beads:

  • Agarose beads
  • Magnetic Agarose beads
  • Magnetic Particles M-270

Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound

Based on the properties of the different beads (see table), we recommend:

  • Agarose beads for very low background and high binding capacity IP
  • Magnetic Agarose beads for magnetic separation and high binding capacity IP
  • Magnetic Particles M-270 for IP of very large proteins/complexes
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Topics: GFP-Trap, Immunoprecipitation, RFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Spot-Trap

GFP-Trap in multiwell plates

Posted by Christoph Eckert on Mar 19, 2020 2:10:56 PM

GFP-Trap®Multiwell Plate for convenient high throughput analysis of protein interactions. It consists of our anti-GFP VHH immobilized in multiwell plates. The GFP-Trap Multiwell Plate is optimized for immunoprecipitation (IP), IP-MS, and ELISA. Here you can find the protocol for sandwich ELISA und immunoprecipitation (IP) with GFP-Trap

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Topics: GFP Immunoprecipitation

5 Tips for Successful Immunoprecipitations (IP)

Posted by Klaus Herick on Mar 3, 2020 3:15:19 PM

The pull-down of proteins is an established technology. It can be difficult, particularly when the protein of interest is expressed at low levels. Here we discuss 5 tips that help you to improve your IP results.

 

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Topics: Immunoprecipitation, GFP Immunoprecipitation

What GFP-Trap should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Jan 20, 2020 4:42:38 PM

ChromoTek GFP-Trap® is optimized for the immunoprecipitation of GFP-tagged proteins and their interacting factors.

The GFP-Trap consists of the ChromoTek anti-GFP Nanobody/ VHH that is coupled to 3 different types of beads or is immobilized in a 96 multiwell plate:

  • Agarose beads
  • Magnetic Agarose beads
  • Magnetic Particles M-270
  • 96 Multiwell Plate

Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound

Based on the properties of the different matrices (see table), we recommend:

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Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody

GFP-Trap Magnetic Particles M-270: Most cited GFP-Nanobody conjugated to Magnetic Particles M-270

Posted by Christoph Eckert on Jan 17, 2020 11:27:30 AM

GFP-Trap® Magnetic Particles M-270 is optimized for the immunoprecipitation (IP) of large GFP-tagged proteins and for Co-IP of protein complexes. It consists of ChromoTek’s established anti-GFP Nanobody conjugated to Magnetic Particles M-270. Hence, also GFP-Trap Magnetic Particles M-270 has the very high affinity of 1 pM like GFP-Trap Agarose and Magnetic Agarose.

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Topics: GFP Immunoprecipitation, GFP Nanobody

Which are 2018's most used Antibodies?

Posted by Klaus Herick on Aug 26, 2019 4:29:03 PM

 
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Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody

Working with green fluorescent proteins: Tools and properties

Posted by Christoph Eckert on Feb 7, 2019 1:18:14 PM

Jellyfish Green Fluorescent Protein (GFP) and its derivatives are still the most frequently used fluorescent proteins in biomedical research. Recently, additional green fluorescent proteins have been discovered in higher animals such as crustaceans and lancelets. These FPs share a common fold, but diverge widely in their primary sequence. Thus, they require novel, dedicated antibody research tools. Here is an overview about EGFP (the most commonly used GFP derivative), TurboGFP and mNeonGreen.

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Topics: Nanobody, mNeonGreen, mNeonGreen immunoprecipitation, GFP, GFP Immunoprecipitation, TurboGFP, TurboGFP immunoprecipitation, EGFP

On-bead digestion protocol: From immunoprecipitation with nanobodies to MS analysis

Posted by Christoph Eckert on Sep 15, 2017 3:52:48 PM

What makes on-bead digestion favorable?

Just pull down your protein of interest with immobilized nanobodies, also termed VHHs or single domain antibodies. Then follow the on-bead digestion protocol (see below) and submit the digest to your core facility for effective mass spectrometer analysis of (co-) precipitated proteins.

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Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody, on-bead digestion, GFP mass spec

The best anti-GFP antibody for immunoprecipitation: GFP-Trap

Posted by Christoph Eckert on Sep 7, 2017 1:05:34 PM

Life science laboratories apply green fluorescent proteins (GFP) to study protein localization, interaction and dynamics in fluorescence microscopy. Immunoprecipitation (IP), mass spectrometry (MS), co-immunoprecipitation (Co-IP) and/or affinity purification investigate more aspects including posttranslational modifications (PTMs), DNA binding, and protein-protein interaction. Here, we compare two different antibody systems for immunoprecipitation of GFP-fusion proteins: GFP-Trap and anti-GFP IgG antibody

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Topics: GFP-Trap, GFP, GFP Immunoprecipitation, Best GFP antibody for IP, Best antibody for immunoprecipitation, GFP VHH, GFP Nanobody

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