Red Fluorescent Protein (RFP), isolated from sea anemones, is a molecular tag commonly used for studying protein localization, reporter gene expression, and protein-protein interactions. Anti-RFP Agarose Resin is an agarose-based matrix coupled with a recombinant anti-RFP nanobody. It can be used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and affinity chromatography of native RFP, RFP mutants, and their fusion proteins.
This product features:
(1) The antibody exhibits extremely high affinity for RFP, enabling efficient capture of RFP-tagged proteins from lysates.
(2) Cleaner backgrounds in SDS-PAGE detection after immunoprecipitation, which is more conducive to identifying protein interactions.
(3) The agarose base matrix offers low non-specific adsorption and good compatibility. With a binding capacity greater than 1 mg per mL medium (can reach up to 5 mg/mL for RFP), it is suitable for large-scale purification of RFP fusion proteins.
Aladdin Anti-RFP Agarose Resin is stored in a solution containing 0.1% ProClin 300, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Specification
|
Matrix
| Highly cross-linked 4% Agarose Microspheres
|
Ligand
| Anti-RFP Nanobody
|
Particle Size Range
| 45~165 μm
|
Binding Capacity
| >1 mg RFP-tagged protein / mL Resin
|
Max Pressure
| 0.3 MPa, 3 bar
|
Reagent Tolerance
| Stable up to 80℃, 1mM DTT, 3M Guanidinium•HCl, 8M Urea, 2M NaCl, 2% Nonidet P40 Substitute, 1% SDS, 1% Triton X-100
|
Storage
| 0.1% ProClin 300, 2-8℃
|
Shelf Life
| 2 years |
Instructions for Use
1. Sample Preparation
Prior to column loading, ensure the sample solution has appropriate ionic strength and pH. The sample or cell culture medium can be diluted with equilibration buffer or dialyzed against it.
It is recommended to centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane before application to reduce impurities, improve purification efficiency, and prevent column clogging.
2. Buffer Preparation
It is recommended to filter all water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Equilibration/Wash Buffer: 50 mM Tris, 0.15 M NaCl, pH 7.4
Acidic Elution Buffer: 0.1 M Glycine-HCl, pH 3.0
Neutralization Buffer: 1 M Tris-HCl, pH 8.0
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-RFP Agarose Resin into a suitable chromatography column. Equilibrate with 5 column volumes (CV) of equilibration buffer.
(2) Apply the sample to the equilibrated medium. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10-20 CV of wash buffer to remove non-specifically adsorbed impurities. Collect the wash fraction.
(4) Acidic Elution: Elute with 5 CV of acidic elution buffer. Add a volume of neutralization buffer equal to one-tenth of the elution volume to the collected fractions to adjust the pH to 7.0-8.0. Collect fractions separately.
*Note: After acidic elution, immediately re-equilibrate the medium with equilibration buffer. Do not leave the Anti-RFP Agarose Resin in the elution buffer for more than 20 minutes.*
(5) Wash the column with 3 CV of elution buffer, then re-equilibrate with equilibration buffer until neutral pH is reached.
(6) Equilibrate with 3 CV of storage buffer. Store the medium at 2-8°C.
3.2 Batch/Binding Adsorption
(1) Medium Preparation: Transfer an appropriate amount of Anti-RFP Agarose Resin to a column or tube and allow the storage solution to drain. Wash with 5 CV of equilibration buffer.
(2) Add the sample solution and incubate with shaking or gentle mixing at 4°C or room temperature for at least 30 minutes (do not use a magnetic stirrer).
(3) After incubation, centrifuge the mixture (5000 × g, 1 min) or filter to collect the medium.
(4) Transfer the medium to a column. Wash with equilibration buffer until the UV baseline stabilizes.
(5) Elute using the acidic elution buffer.
(6) Medium regeneration and storage are the same as described in sections 3.1 (5) and (6).
3.3 Immunoprecipitation (IP) Protocol
(1) Medium Preparation: Add 40 µL of the Anti-RFP Agarose Resin slurry (approx. 20 µL settled medium volume) to a 1.5 mL microcentrifuge tube. Centrifuge at 5000 × g for 1 min. Carefully remove and discard the supernatant.
(2) Medium Equilibration: Add 0.5 mL of equilibration buffer to resuspend the medium. Centrifuge at 5000 × g for 1 min. Discard the supernatant. Repeat this step once.
(3) Sample Binding: Add 200-1000 µL of sample to the prepared medium. Mix thoroughly. Incubate the tube at room temperature on a rotator/mixer for gentle inversion for at least 1 hour (For proteins prone to degradation, the use of protease inhibitors and performing the procedure at 2-8°C in a cold room or chromatography refrigerator is recommended). Centrifuge at 5000 × g for 1 min. Discard the supernatant, being careful not to disturb the medium pellet.
(4) Washing: Add 0.5 mL of wash buffer to resuspend the medium. Mix gently. Centrifuge at 5000 × g for 1 min. Discard the supernatant. Repeat this washing step three more times.
(5) Sample Elution: Choose the elution method based on downstream application needs.
* A. Acidic Elution: Add 100 µL of acidic elution buffer to resuspend the medium. Incubate at room temperature for 5 min. Centrifuge at 5000 × g for 1 min. Carefully collect the supernatant without disturbing the medium. Neutralize the eluate immediately using neutralization buffer. Store eluted samples at 4°C; for long-term storage, keep at -20°C.
* B. Denaturing Elution: Conventional protein loading buffer contains β-mercaptoethanol or DTT, which reduces disulfide bonds, and SDS, which denatures proteins. This method will elute the target protein but also denature the ligand (nanobody), making the medium unsuitable for reuse.
Add 20 µL of 2× Loading Buffer to the medium. Heat at 95°C for 5 min. Centrifuge at 5000 × g for 1 min. Carefully collect the supernatant for SDS-PAGE analysis.
Troubleshooting
