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GFP-Trap® Magnetic Agarose Kit

AM, ChIP, EAA, IP, MS, Co-IP, Pull-Down, Purif Reactivity: Aequorea victoria Cell Extracts
Pubmed (932)
Catalog No. ABIN1889488
$540.00
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  • Target
    Antibody Type
    Recombinant Antibody
    Reactivity
    Aequorea victoria
    Host
    Camelid (Camelidae)
    Conjugate
    Magnetic Agarose Beads
    Application
    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)
    Purpose
    GFP-Trap® is a high quality GFP-binding protein coupled to a monovalent matrix (magnetic agarose beads) for biochemical analysis of GFP fusion proteins and their interacting partners.
    Brand
    GFP-Trap®
    Sample Type
    Cell Extracts
    Specificity
    Binding capacity: 10 µl GFP-Trap®_MA slurry binds 3 - 4 µg of GFP
    Cross-Reactivity (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.
    Characteristics
    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.
    Components
    GFP-Trap® coupled to magnetic agarose beads
    Material not included
    Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
  • Application Notes
    Green fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass pectroscopy 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.
    Comment

    Particle size ~ 40 µm

    Assay Time
    1.5 h
    Protocol
    • 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)
    Reagent Preparation

    Suggested buffer composition

    • Lysis buffer (CoIP): 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA,0.5% NP-40
    • 10x RIPA buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate
    • Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
    • Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
    • Elution buffer: 200 mM glycine pH 2.5

    Sample Collection
    Harvest cells:
    For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) expressing a GFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 ml ice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4°C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.

    Lyse cells
    • 1. Resuspend cell pellet in 200 µL ice-cold lysis buffer by pipetting or using a syringe.
      note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included). optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).
    • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
    • 3. Centrifuge cell lysate at 20.000x g for 10 min at +4°C. Transfer lysate to a pre-cooled tube. Add 300 µl dilution buffer to lysate. Discard pellet.
      note: At this point cell lysate may be put at -80°C for long-term storage. optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer

    We recommend that during incubation with the beads the final concentration of detergents does not exceed 0.2% to avoid unspecific binding to the matrix. If required, use more dilution buffer to dilute the supernatant accordingly.
    Assay Procedure

    Equilibrate beads

    • 4. Vortex GFP-Trap®_MA beads and pipette 25 µL bead slurry into 500 µL ice-cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.

    Bind proteins:
    • 5. Add diluted lysate (step 3) to equilibrated GFP-Trap®_MA beads (step 4). If required, save 50 µL of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4°C.
    • 6. Magnetically separate beads until supernatant is clear. If required, save 50 µL supernatant for immunoblot analysis. Discard remaining supernatant.

    Wash beads:
    • 7. Resuspend GFP-Trap®_MA beads in 500 µL dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
      optional: Increase salt concentration in the second washing step up to 500 mM.

    Elute proteins:
    • 8. Resuspend GFP-Trap®_MA beads in 100 µL 2x SDS-sample buffer.
    • 9. Boil resuspended GFP-Trap®_MA beads for 10 min at 95°C to dissociate immunocomplexes from GFP-Trap®_MA beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant.
    • 10. optional instead of steps 8 and 9: elute bound proteins by adding 50 µl 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by magnetic separation. Transfer the supernatant to a new tube and add 5 µL 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.

    Restrictions
    For Research Use only
  • Buffer
    Storage buffer: 20 % EtOH
    Handling Advice
    Do not freeze.
    Storage
    4 °C
    Expiry Date
    12 months
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  • Target Name (Antigen)
    Alternative Name
    GFP
    Synonyms
    green fluorescent protein, gfp
    Background
    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 .
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