Green fluorescent protein is widely used in protein localization and protein dynamics analysis. GFP-beads are conjugated by GFP-tag antibody and agarose gel microspheres, specifically designed for purifying and detecting GFP-tagged proteins in cell lysates or bacterial lysates. It is suitable for IP experiments. When the cell or bacterial lysate is mixed with this reagent, the GFP-tagged recombinant protein in the sample specifically binds to GFP beads, while other contaminants do not. This reagent has a stable structure and high efficiency in binding to GFP-tagged proteins. The IP products can be applied in biochemical analysis methods such as mass spectrometry analysis and enzyme activity determination. This product is a specially prepared GFP beads product. When using this product for IP experiments, in the subsequent IP-WB steps, the appearance of bands from the light and heavy chains of the antibody can be completely avoided.

IP

All Species Expected

Transfected Only

Anti-GFP Tag mAb

IP 1:50

Mouse IgG

The beads are recommended to be stored at 4℃. The product is stable for 12 months at 4℃. Freezing the magnetic beads will irreversibly damage the bead structure.

Lysis buffer (IP、CoIP)：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA;0.5% NP-40

RIPA buffer：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA;0.1% SDS;1% Triton X-100;1% Deoxycholate

Dilution/Wash buffer：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA

2×SDS loading buffer：120mM Tris-HCl pH 6.8; 20% glycerol; 4% SDS, 0.04% bromophenol blue; 10%β-mercaptoethanol

AB_2936244

Experimental Procedure for Label Antibody-Conjugated Agarose

1. Experimental Preparation

Use 1.5mL microcentrifuge tubes or spin columns as experimental vessels. 

Vortex or invert the tube or vial containing Anti-GFP agarose thoroughly to achieve complete resuspension of the beads.

2. Sample and Agarose Incubation 

Using a wide-bore pipette tip, add 20–50μL of 50% (v/v) agarose suspension to the cell lysate.
Note: The lysis buffer (10mM Tris-HCl, pH7.5; 150mM NaCl; 0.5mM EDTA; 0.5% NP-40) should be freshly prepared. For samples expressing high levels of tagged protein, 200μL of lysate (containing 200-500μg total protein) typically yields optimal immunoprecipitation (IP) results. It is recommended to include a protease inhibitor cocktail (e.g., catalog no. A10004: 200×Protease Inhibitor Cocktail) in the lysis buffer.

3. Binding Incubation 

Incubate the mixture gently on a rotator or shaker at 4℃ for 3-4 hours or overnight to ensure efficient binding between the antibody and the target protein.

4. Agarose Bead Washing 

Wash the agarose beads with 1 mL of wash buffer (10mM Tris-HCl, pH7.5; 150mM NaCl; 0.5mM EDTA; 1% NP-40; 0.5% sodium deoxycholate). Alternatively, 1×PBST (0.05% Tween-20) may be used.

Centrifuge at 2000×g for 3 minutes, carefully remove the supernatant, and repeat the washing step three times. Avoid disturbing or losing the agarose bead pellet during aspiration.

5. Target Protein Elution (Choose One of the Following Methods)

Option 1: Acid Elution
Add 30–50μL of acidic elution buffer to the washed beads and mix gently by tapping. Immediately centrifuge at 2000×g for 3 minutes and transfer the supernatant to a fresh tube, taking care not to carry over any agarose beads.

Note: Neutralize the eluate immediately by adding 1μL of 1.5 M Tris-HCl (pH 9.0) per 20 μL of elution buffer.

Option 2: SDS Loading Buffer Elution 
Add 30μL of 2×SDS-PAGE loading buffer to the beads and mix thoroughly. Heat the sample at 100°C for 5 minutes to elute bound proteins. Centrifuge at 2000×g for 3 minutes and transfer the supernatant to a new tube.
Note: This method results in co-elution of the target protein along with antibody heavy and light chains (IgG).

6. Downstream Applications     

Proceed with SDS-PAGE and Western blotting, or other appropriate analytical methods, for detection and analysis of the eluted protein.