ChromoBlog

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.

ChromoTek's Nano-Traps are optimized for the immunoprecipitation (IP) of proteins and their interacting factors. Nano-Traps comprise of a Nanobody/ V_HH 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
  

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

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.

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/ V_HH 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:

• GFP-Trap Agarose for very low background and high binding capacity IP
• GFP-Trap Magnetic Agarose for magnetic separation and high binding capacity IP
• GFP-Trap Magnetic Particles M-270 for IP of very large proteins/complexes
• GFP-Trap Multiwell Plates for high throughput analysis

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.

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.

What makes on-bead digestion favorable?

Just pull down your protein of interest with immobilized nanobodies, also termed V_HHs 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.

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