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    Anti-GFP tag Mouse mAb conjugated Agarose Beads

    Catalog number :AT0077
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    Since the detection of intracellular Aequorea victoria Green Fluorescent Protein (GFP) requires only irradiation by UV or blue light, it provides an excellent means for monitoring gene expression and protein localization in living cells. Agarose beads conjugated anti-GFP Mouse mAb can detect GFP fusion protein on immunoprecipitation.
    Overview
    Description
    Anti-GFP tag Mouse mAb conjugated Agarose Beads
    Reactivity
    This antibody reacts with GFP fusion protein on Immunoprecipitation (IP) and Co-IP. It reacts with GFP, EGFP, YFP, CFP, AcGFP, Venus and Citrine.
    Tested applications
    IP and Co-IP
    Properties
    Immunogen
    Recombinant A. victoria GFP protein
    Isotype
    Mouse mAb
    Form
    provided as 1ml of a 50% slurry suspended in PBS containing preservative (0.09% sodium azide).
    *Azide may react with copper or lead in plumbing system to form explosive metal azides. Therefore, always flush plenty of water when disposing materials containing azide into drain.
    Storage instruction
    4°C
    Host
    Mouse
    Applications
    Protocols
    Protocol for Immunoprecipitation of GFP-Fusion Proteins from mammalian cell extract
     
    1. Harvest cells
    1). For one immunoprecipitation reaction the use of ~106 - 107 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. After washing:
     
     
    2. 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 20000x 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.
     
    3. Equilibrate beads
    1). Vortex anti-GFP agarose beads and pipette 25 μl bead slurry into 500 μl ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.
     
    4. Bind proteins
    1). Add diluted lysate (step 2.3) to equilibrated anti-GFP agarose beads (step 3.1). If required, save 50 μl of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4°C.
     
    2). Centrifuge at 2500x g for 2 min at +4°C. If required, save 50 μl supernatant for immunoblot analysis. Discard remaining supernatant.
     
    5. Wash beads
    1). Resuspend anti-GFP agarose beads in 500 μl ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.
    optional: Increase salt concentration in the second washing step up to 500 mM.
     
    6. Elute proteins
    1). Resuspend anti-GFP agarose beads in 100 μl 2x SDS-sample buffer.
     
    2). Boil resuspended anti-GFP agarose beads for 10 min at 95°C to dissociate immunocomplexes from anti-GFP agarose beads. anti-GFP agarose beads can be collected by centrifugation at 2500x g for 2 min at 4°C and SDS-PAGE is performed with the supernatant.
     
    3). optional instead of steps 6.1 and 6.2: 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 new tube and add 5 μl 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.

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