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.
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
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.
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
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
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
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Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody
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
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
Topics: GFP-Trap, GFP, GFP Immunoprecipitation, Best GFP antibody for IP, Best antibody for immunoprecipitation, GFP VHH, GFP Nanobody
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.
The PreOmics iST kit can also be used with other ChromoTek Nano-Traps.
Topics: GFP-Trap, Immunoprecipitation, Mass spec, GFP Immunoprecipitation, GFP MS
