GFP Tag Magrose Beads

• Rapid magnetic responsiveness
• Excellent microsphere dispersibility
• Extremely low nonspecific adsorption
• Abundant binding sites

• Detection and purification of GFP, EGFP, and their fusion proteins
• Immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) experiments

I.Buffer Preparation

The following are commonly used buffer compositions suitable for most GFP fusion protein purification applications. Adjustments can be made as needed. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm membrane for sterilization before use.

1)Binding/Washing Buffer: 50 mM Tris，0.15 M NaCl，pH7.4.

2)Elution Buffer: 0.1 M Glycine-HCl, pH3.0.

3)Neutralization Buffer: 1.0 M Tris-HCl, pH 8.0.

II.Purification of GFP-Tagged Proteins

1.Magnetic Bead Pretreatment

1)Vortex the GFP Tag Magrose Beads thoroughly to ensure complete mixing. Use a pipette to transfer an appropriate volume of bead suspension into a centrifuge tube. Place the tube in a magnetic separator and let it stand for 1 minute to allow magnetic separation. Carefully remove the supernatant and take the tube out of the separator.

Note: The volume of bead suspension should be calculated based on the sample volume and protein content.

2)Add an equal volume of Binding/Washing Buffer to the centrifuge tube. Vortex to resuspend the beads, then perform magnetic separation and discard the supernatant. Repeat the washing step twice.

Note: To minimize bead loss during magnetic separation, once the solution becomes clear, tightly close the tube cap and keep the tube on the magnetic separator. Then, while holding both the magnetic separator and the tube, invert them together several times to rinse any residual beads from the tube cap using the clear solution. Let it stand briefly until the solution clears again before proceeding. This procedure should be followed for all subsequent magnetic separation steps.

2.Binding of Magnetic Beads to Target Protein

1)Add the sample solution to the pretreated magnetic beads, then place the centrifuge tube on a vortex mixer and vortex for 15 seconds.

2)Place the tube on a rotator and mix at room temperature for at least 30 minutes. Alternatively, to prevent degradation of the target protein, rotate at 2–8 °C for 1–2 hours.

3)Perform magnetic separation and transfer the supernatant to a new centrifuge tube for downstream analysis. Remove the tube from the separator and proceed with the subsequent washing steps.

3.Washing of Magnetic Beads

1)Add 5 volumes of Binding/Washing Buffer to the centrifuge tube containing the magnetic beads. Rotate and mix for 2 minutes to resuspend the beads, then perform magnetic separation. Transfer the Washing Buffer to a new centrifuge tube for sampling and analysis. Repeat this washing step once.

2)To avoid contamination of the target protein by nonspecifically bound proteins on the wall of the original tube, add Binding/Washing Buffer to resuspend the beads, then transfer the bead suspension to a new centrifuge tube. Perform magnetic separation and transfer the supernatant to the wash collection tube.

4.Target Protein Elution

Two elution methods are provided below. Choose the appropriate method based on your downstream analysis.

1)Denaturing Elution: Suitable for SDS-PAGE analysis. Add SDS-PAGE Loading Buffer (user-supplied) to the EP tube, mix well, and heat at 95 °C for 5 minutes. Then perform magnetic separation, and collect the supernatant for SDS-PAGE.

Note: GFP Tag Magrose Beads cannot be reused after denaturing elution.

2)Non-denaturing elution: Adjust the elution volume as needed to achieve the desired concentration of the target protein. Add 3–5 volumes of Elution Buffer, rotate and mix at room temperature for 5–10 minutes to resuspend the beads, then perform magnetic separation. Collect the elution into a new EP tube to obtain the target protein. Add Neutralization Buffer equal to one-tenth of the elution volume to adjust the pH to 7.0–8.0.

Note: After acidic elution, immediately equilibrate the magnetic beads with Binding/Washing Buffer. GFP Tag Magrose Beads should not remain in the elution buffer for more than 20 minutes.

III.IP/Co-IP Assay

1. Magnetic Bead Pretreatment Same as in protein purification.

2. Binding of Magnetic Beads to Bait Protein

1)Add the GFP-tagged bait protein solution to the pretreated magnetic beads and gently invert to mix. Place the centrifuge tube on a rotator and mix at room temperature for 30 minutes. Alternatively, to prevent protein degradation, rotate at 2–8 °C for 1–2 hours.

2)Perform magnetic separation and transfer the supernatant to a new centrifuge tube for downstream analysis. Add 5 volumes of Binding/Washing Buffer to the tube containing the beads, mix for 2 minutes to resuspend the beads, and perform magnetic separation. Repeat the washing step twice to obtain the bait protein–magnetic bead complex.

Note: If performing an IP assay, skip Step 3 and proceed directly to Step 4. If performing a Co-IP assay, continue with Step 3.

3. Binding of Target Protein to the Complex

1)Add the lysate containing the target protein to the GFP fusion protein–magnetic bead complex prepared in the previous step. Place the centrifuge tube on a rotator and mix at room temperature for at least 30 minutes.

2)Perform magnetic separation and transfer the supernatant to a new centrifuge tube for downstream analysis. Add 5 volumes of Binding/Washing Buffer to the bead-containing tube, mix for 2 minutes to resuspend the beads, and perform magnetic separation. Repeat the washing step twice to obtain the final target protein–bait protein–magnetic bead complex.

4. Target Protein Elution

Two elution methods are provided below. Choose the appropriate method based on your downstream analysis.

1)Denaturing Elution: Suitable for SDS-PAGE analysis. Add SDS-PAGE Loading Buffer (user-supplied) to the EP tube, mix well, and heat at 95 °C for 5 minutes. Then perform magnetic separation, and collect the supernatant for SDS-PAGE.

Note: GFP Tag Magrose Beads cannot be reused after denaturing elution.

2)Non-denaturing elution: Adjust the elution volume as needed to achieve the desired concentration of the target protein. Add 3–5 volumes of Elution Buffer, rotate and mix at room temperature for 5–10 minutes to resuspend the beads, then perform magnetic separation. Collect the elution into a new EP tube to obtain the target protein. Add Neutralization Buffer equal to one-tenth of the elution volume to adjust the pH to 7.0–8.0.

Note: After acidic elution, immediately equilibrate the magnetic beads with Binding/Washing Buffer. GFP Tag Magrose Beads should not remain in the elution buffer for more than 20 minutes.

Store at 4℃ for 2 years

1.Avoid freezing, drying, or subjecting the magnetic beads to high-speed centrifugation.

2.To minimize bead loss, magnetic separation time should be no less than 1 minute each time.

3.Before withdrawing beads from the storage tube, ensure thorough resuspension by vortexing. Avoid bubble formation during handling.

4.It is recommended to use high-quality pipette tips and reaction tubes to reduce bead and solution loss due to surface adhesion.

5.If the solution is too viscous to resuspend the magnetic beads by inverting the tube, pipetting up and down or briefly vortexing can be used to fully resuspend the beads.

6.Users may retain the supernatant removed after magnetic separation for sampling and analysis, as needed, to evaluate the purification process and optimize the protein purification workflow.

7.This product can be reused. When reusing magnetic beads, it is recommended to purify the same type of protein. For purification of different proteins, using fresh beads is advised.

8.This product is intended for use by qualified professionals for scientific research only. It is not for clinical diagnosis or treatment, not for use in food or pharmaceuticals, and must not be stored in residential areas.

9.For your safety and health, please wear a lab coat and disposable gloves during operation.