Anti-RFP Affinity Gel

Recommended Buffers (Not Provided)

Note: Prepare all buffers with ultrapure water and sterilize them by filtration through a 0.45 μm or 0.22 μm membrane.

Protein Purification

Before loading, centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane to reduce impurities and improve purification efficiency.

Medium-Pressure Chromatography Column Method

1. Column Packing: Pack the Anti-RFP Affinity Gel into a suitable chromatography column and connect it to the chromatography system.

2. Column Equilibration: Equilibrate with 5 column volumes of binding/wash buffer. Repeat 2-3 times.

3. Sample Loading: Load the sample using a pump or sample loop and collect the flow-through. Repeated loading may improve binding efficiency.

Notes: a. Select the gel volume according to the amount of target protein and do not exceed the binding capacity;
b. Increased sample viscosity or volume may increase column back pressure.

4. Washing: Wash with 10-20 column volumes of binding/wash buffer and collect wash fractions until the UV absorbance baseline becomes stable.

5. Elution: Elute with 3-5 column volumes of acidic elution buffer, collect fractions, and immediately add neutralization buffer at 1/10 of the total eluate volume.

Note: Acid-eluted proteins may be stored at 4°C for a short period or at -20°C for long-term storage.

6. Regeneration: Continue washing with 5-10 column volumes of acidic elution buffer to remove residual protein, then equilibrate to neutral pH with binding/wash buffer. Do not expose the gel to acidic elution buffer for more than 20 min.

7. Storage: Equilibrate with 5-10 column volumes of storage buffer and store at 2-8°C.

Gravity-Flow Column Method

1. Column Packing: Transfer an appropriate volume of Anti-RFP Affinity Gel suspension into a gravity-flow column and remove the storage solution.

2. Column Equilibration: Equilibrate with 5 column volumes of binding/wash buffer. Repeat 2-3 times.

3. Sample Loading: Allow the loaded sample to remain in the column for at least 2 min, then collect the flow-through. Repeated loading may improve binding efficiency.

4. Washing: Wash with 10-15 column volumes of binding/wash buffer and collect the wash fractions.

5. Elution: Elute with 3-5 column volumes of acidic elution buffer, collect fractions, and immediately add neutralization buffer at 1/10 of the total eluate volume.

6. Regeneration: Wash with 5-10 column volumes of acidic elution buffer and then equilibrate to neutral pH with binding/wash buffer.

7. Storage: Equilibrate with 5 column volumes of storage buffer and store at 2-8°C.

Centrifugation Method

1. Gel Pretreatment: Transfer an appropriate amount of Anti-RFP Affinity Gel suspension into a centrifuge tube, centrifuge at 5,000 × g for 1 min, discard the supernatant, and wash with 5 gel volumes of binding/wash buffer 2-3 times.

2. Binding: Add the sample and incubate at 4°C for 2-4 h or at 37°C for 0.5-2 h.

3. Washing: Centrifuge at 5,000 × g for 1 min, retain the flow-through if required, and wash with 5 gel volumes of binding/wash buffer 3-5 times.

4. Elution: Add 3-5 gel volumes of elution buffer, incubate at room temperature for 5-10 min, centrifuge at 5,000 × g for 1 min, and collect the supernatant. Immediately neutralize acidic eluates. The elution may be repeated 2-3 times.

5. Regeneration and Storage: Wash sequentially with 5-10 gel volumes of binding/wash buffer, 5-10 gel volumes of ddH₂O, and 2 gel volumes of storage buffer. Store at 2-8°C.

Immunoprecipitation

1. Gel Pretreatment: Resuspend the Anti-RFP Affinity Gel and transfer 40 μL of suspension (approximately 20 μL settled gel) into a 1.5 mL tube. Centrifuge at 5,000 × g for 1 min and wash with 500 μL of binding/wash buffer 3-4 times.

2. Sample Binding: Add 200-1,000 μL of sample and incubate on a rotator at 4°C for 2 h or overnight. Centrifuge at 5,000 × g for 1 min and transfer the supernatant to a new tube.

3. Washing: Wash with 500 μL of binding/wash buffer at least three times, or until the OD₂₈₀ of the wash supernatant is below 0.05.

4. Elution: Select acidic or denaturing elution according to downstream needs. For acidic elution, add 50-100 μL of elution buffer, incubate for 5 min, centrifuge, and immediately neutralize. For denaturing elution, add 20-50 μL of 2× SDS-PAGE Loading Buffer, heat at 95°C for 5 min, centrifuge, and collect the supernatant.

Note: After denaturing elution with SDS, β-mercaptoethanol, or DTT, the immobilized antibody or ligand may be inactivated and the gel should not be reused.