Introduction:

Anti-GFP Magnetic Beads are 200 nm nano-sized magnetic beads with a surface densely coated with high-quality mouse monoclonal anti-Myc antibody. The nano-magnetic beads provide an ultra-large specific surface area, offering more binding sites and thus requiring less magnetic bead volume. They exhibit minimal non-specific binding in cell lysates, serum, plasma, and other samples.

This product is widely used for immunoprecipitation (IP) or co-immunoprecipitation (Co-IP) experiments targeting GFP-tagged fusion proteins. Due to the use of magnetic separation, each IP and Co-IP experiment can save approximately 40% of the time.

Information：

Storage: Store at 2-8 ℃. Valid for 24 months.

Notes:

1. Please avoid high-speed centrifugation, drying, or freezing the magnetic beads, as these operations will cause bead aggregation and reduce binding capacity.
2. Different types of antibodies and targets have varying binding specificities. Antibody-target binding is also affected by the Lysis/Wash Buffer. Therefore, optimization details can be adjusted based on actual results, such as customizing buffers.
3. Ensure thorough mixing of magnetic beads with the sample solution to prevent drying.
4. For scientific research use by professionals only. Not for clinical diagnosis or treatment. Not for use in food or drugs. Do not store in residential areas.
5. For your safety and health, please wear disposable gloves during operation.

Protocol (for reference only):

Note: Lysis buffer can be prepared manually or directly purchased as an IP Grade kit.

A. Adherent Cell Samples

1. Remove the culture medium and wash the cells twice with PBS.
2. Collect the cells into a 1.5mL EP tube. Add IP Lysate/Wash Buffer according to the proportion table below, along with appropriate inhibitors (e.g., PMSF). Mix well and place on ice for 5-20 minutes (depending on the number of cells).
3. Centrifuge at 4℃, 12000-16000xg for 10 minutes. Transfer the supernatant to a new tube for subsequent IP experiments (or store at -80℃ long-term).

B. Suspension Cell Samples

1. Centrifuge the cell suspension (4℃, 500-1000xg, 10min) and discard the supernatant.
2. Wash the cell pellet once with 1 × PBS, resuspend the cell pellet in PBS, (4℃, 500-1000xg, 5 min), collect the cells, and discard the supernatant.
3. Resuspend the cells in ice-cold IP Lysis/Wash Buffer. Use 500 µL of IP Lysis/Wash Buffer per 50 mg of cells. Simultaneously add protease inhibitors such as PMSF, mix well, and place on ice for 5-20 min (invert several times during this period).
4. Centrifuge to collect the supernatant (4℃, 12000-16000xg, 10 min). Place the supernatant on ice for subsequent experiments (or store at -80℃ for long-term storage).

C. Serum Samples

It is generally recommended to dilute the serum sample to a target protein concentration of 50-150µg/mL using IP Lysate/Wash Buffer. Place on ice for use or store at -20℃ (avoid repeated freeze-thaw cycles).

Preparation of Immune Complexes

Note: The amount of sample, antibody, and incubation time depend on each specific antibody-antigen system. Optimization may be required to obtain the maximum yield.

The following experimental protocol is for 2 - 10 μg of cell lysate and purified immunoglobulin. The amount needed can be scaled up proportionally.

1. In a microcentrifuge tube, combine cell lysate from each sample with 2-10 μg IP‑grade antibody. Use 500‑1500 μg of total protein per immunoprecipitation reaction.
2. Dilute the antibody (used for IP) and the prepared sample to 300 - 500 μL with IP Lysate/Wash Buffer.
3. Incubate at room temperature for 1 - 2 h, or at 4 ℃ 2-4 h, to form the immune complex.

Immunoprecipitation

Note: To ensure uniform distribution of the magnetic beads, gently invert the tube or vortex briefly to mix the beads.

1. Transfer 20-50 µL of Anti-GFP magnetic beads into a 1.5 mL centrifuge tube.
2. Add 500 μL pre-chilled PBS to the beads and mix gently.
3. Place the centrifuge tube on a magnetic stand to collect beads against one side of the tube. Remove the supernatant.
4. Add 200-500 μL IP Lysis/Wash Buffer to the tube. Invert the tube several times or vortex gently for 1 min. Collect beads with a magnetic stand and discard the supernatant.
5. Add GFP-tagged protein sample to the tube containing magnetic beads. Incubate with mixing for 1-2 h at room temperature, or 2-4 h at 4 ℃.
6. Harvest beads using a magnetic stand. Save the unbound flow-through sample for subsequent analysis.
7. Add 1000 μL IP Lysis/Wash Buffer. Gently mix the beads for 5-10 min. Collect beads and discard supernatant. Repeat this wash step two more times.
8. Two antigen-elution protocols are provided in this manual. Select the appropriate method according to downstream assay requirements.

Denaturing Elution:

Add 80-100 μL of 1 × SDS-PAGE Sample Loading Buffer. Heat the sample at 100  ℃ in a water bath or heat block for 10 min. Separate magnetic beads with a magnetic stand. Retain the supernatant containing target antigen for SDS-PAGE analysis.

Non-denaturing Elution:

Samples eluted by this method retain native biological activity and are suitable for downstream functional assays.

Add 100 μL Elution Buffer. Incubate the tube at room temperature with mixing for 5-10 min. Isolate beads magnetically and keep the supernatant containing target antigen.

Add 20 μL Neutralization Buffer per 100 μL eluate for pH neutralization prior to downstream functional analysis.