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”.
Topics: GFP-Trap, Immunoprecipitation, Co-IP
How to conduct a Co-immunoprecipitation (Co-IP)?
Topics: GFP-Trap, Immunoprecipitation, Co-IP
Overview of ChromoTek’s Nanobody-based tools for cancer research
Cancer is still a leading cause of mortality. Each year 18 million people worldwide get diagnosed with cancer. The current trend is increasing. This makes cancer research more important.
Topics: Chromobodies, GFP-Trap, p53/Mdm2, p53/Mdm4, MK2-Trap, Vimentin
Tips and tricks for immunoprecipitation of low abundant proteins
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.
Topics: GFP-Trap, GFP Immunoprecipitation
Neuropilin-1 joins ACE2 as SARS-CoV-2 gateaway
Using both Proteintech and Chromotek antibodies, Davy and colleagues published a landmark paper in Science late in 2020, detailing how Neuropilin-1 is required for SARS-CoV-2 host cell entry.
Which beads should I use for my immunoprecipitation?
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
Topics: GFP-Trap, Immunoprecipitation, RFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Spot-Trap
How to obtain a low background in immunoprecipitation assays
Introduction
A high background from unspecific binding of proteins is a common problem in immunoprecipitation (IP). In this blog, the origin of background and different optimization strategies are discussed. Furthermore, it is shown how you can achieve a low background when using the ChromoTek GFP-Trap®, a ready to use reagent for IP of a GFP-tagged proteins.
Topics: GFP-Trap, Immunoprecipitation
What GFP-Trap should I use for my immunoprecipitation?
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:
- 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
Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody
Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody
On-bead digestion protocol: From immunoprecipitation with nanobodies to MS analysis
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.
Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody, on-bead digestion, GFP mass spec