GFP-Trap® M Kit

Details zu Produkt Nr. ABIN509406, Anbieter: Anmelden zum Anzeigen
  • green fluorescent protein
  • gfp
Aequorea victoria
Camelid (Camelidae)
Recombinant Antibody
Magnetic Particles
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
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Verwendungszweck GFP-Trap® is a high quality GFP-binding protein coupled to a monovalent matrix (magnetic particles) for biochemical analysis of GFP fusion proteins and their interacting partners.
Marke GFP-Trap®
Proben Cell Extracts
Spezifität Binding capacity: 10 µL GFP-Trap®_M slurry binds 0.25 - 0.5 µg of GFP
Kreuzreaktivität (Details) GFP-Trap® specifically binds to eGFP, wtGFP, GFP S65T, TagGFP, eYFP, YFP, Venus, Citrin, CFP. No binding to proteins derived from DsRed, all RFPs and TurboGFP can be detected.
Produktmerkmale Antibodies - extremely powerful tools in biomedical research - are large complex molecules (~ 150 kDa) consisting of two heavy and two light chains. Due to their complex structure, the use of antibodies is often limited and hindered by batch-to-batch variations.

Camelidae (camels, dromedaries, llamas and alpacas) possess functional antibodies devoid of light chains, so-called heavy chain antibodies (hcAbs). hcAbs recognize and bind their antigens via a single variable domain (VHH). These VHH domains are the smallest intact antigen binding fragments (~ 13 kDa).

Nano-Traps are based on single domain antibody fragments (VHHs) derived from alpaca.
Bestandteile GFP-Trap® coupled to magnetic particles
The kit is including lysis, wash and elution buffers.
Andere Bezeichnung GFP
Hintergrund The green fluorescent protein (GFP) and variants thereof are widely used to study the subcellular localization and dynamics of proteins. GFP fusion proteins can be expressed in different cell types at different expression levels by transient or stable transfection. Transient expression may provide quick informative results, however, in many cases it is necessary to generate stable cell lines that express the GFP fusion protein of interest at a level similar to the one of the endogenous protein. Quantification of GFP fusion proteins in cells can be tricky since existing methods, like fluorescence microscopy or Western Blotting, are often shows insufficient signal to noise ratios or high signal variabilities .
Applikations-hinweise Green fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass spectroscopy and enzyme activity measurements these GFP-fusion proteins and their interacting factors can be isolated fast and efficiently (one step) via Immunoprecipitation using the GFP-Trap®. The GFP-Trap®_A enables purification of any protein of interest fused to GFP.

Bead size 0.5 - 1 µm

Testdauer 1.5 h
  • Robust and versatile tool for biochemical analyses of GFP-fusion proteins
  • Short incubation times (5 - 30 min)
  • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
  • Low unspecific binding
  • No contaminating heavy and light chains of conventional antibodies
  • Applicable in Chromatin Immunoprecipitation (ChIP)

Before you start: Add 1ml PBS to your cells and scrape them off the petri dish.Transfer to precooled tube, spin 3 min at 500 x g and discard supernatant. Wash cell pellet twice with ice cold PBS, briefly resuspending the cells.

  • 1. For one immunoprecipitation reaction resuspend cell pellet (~10^7 mammalian cells) in 200 µL lysis buffer by pipetting (or using a syringe).
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to lysis buffer
    optional for nuclear/chromatin proteins: add 1 mg/ml DNase and 2.5 mM MgCl2 (not included) to lysis buffer
  • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
  • 3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C.
  • 4. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500 µL – 1000 µL. Discard pellet.
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to dilution buffer
    note: the cell lysate can be frozen at this point for long-term storage at -80°C. For immunoblot analysis dilute 50 µL cell lysate with 50 µL 2x SDS-sample buffer(à refer to as input).
  • 5. Equilibrate GFP-Trap®_M beads in dilution buffer. Resuspend magnetic beads by vortexing and transfer 20 - 30 µL bead slurry in 500 µL ice cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and wash beads 2 more times with 500 µL ice cold dilution buffer.
  • 6. Add cell lysate to equilibrated GFP-Trap®_M beads and incubate the GFPTrap®_M beads with the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C.
    note: during incubation of protein sample with the GFP-Trap®_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix.
  • 7. Magnetically separate beads until supernatant is clear. For western blot analysis dilute 50 µL supernatant with 50 µL 2x SDS-sample buffer (à refer to as nonbound). Discard remaining supernatant.
  • 8. Wash beads three times with 500 µL ice cold wash buffer. After the last wash step, transfer beads to new tube.
    optional: increase salt concentration in the second washing step up to 500 mM
  • 9. Resuspend GFP-Trap®_M beads in 100 µL 2x SDS-Sample buffer or go to step 11.
  • 10. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant (à refer to as bound).
  • 11. optional: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a fresh cup and add 5 µL 1M Tris base (pH 10.4) for neutralization. To increase elution efficiency this step can be repeated.

Beschränkungen Nur für Forschungszwecke einsetzbar
Konzentration 500 µL resin
Buffer 1 x PBS,0.01% Sodium azide
Konservierungs-mittel Sodium azide
Vorsichtsmaßnahmen This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handhabung Do not freeze.
Lagerung 4 °C
Haltbarkeit 12 months
Bilder des Herstellers
 image for GFP-Trap® M Kit (ABIN509406) Left (IP): Pulldown of GFP with GFP-Trap®_A and GFP-Trap®_M from 293T cell extracts. ...
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