Anti-YFP Affinity Gel
Based on 1 Customer Validation
MCE Anti-YFP Affinity Gel can be used for the detection and purification of proteins tagged with GFP and EGFP and IP assays.
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Storage :
4℃, 2 years
Do not dry or freeze
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
The Yellow Fluorescent Protein (YFP), derived from Aequorea Victoria, is a mutant variant (T203Y) of Green Fluorescent Protein (GFP). This mutation shifts its fluorescence emission towards the red spectrum, emitting yellow fluorescence. YFP has been extensively utilized as a reporter gene in the fields of cell biology and molecular biology.
MCE Anti-YFP Affinity Gel is produced through the covalent coupling of high-quality YFP antibody with agarose. It boasts a high loading capacity, exceptional specificity, and stability. This gel can be used for the detection and purification YFP and its fusion-expressed proteins. It is also effective in binding and purifying proteins tagged with GFP and EGFP, however, it does not bind to proteins tagged with BFP or RFP. Moreover, it can be utilized for immunoprecipitation (IP) assays.
The specifications of the product correspond to the actual resin volume, with the resin content of 50%.
Protocol
Recommended Buffers (Not Provided)
| Buffer | Composition |
|---|---|
| Binding/Wash Buffer | 50 mM Tris, 0.15 M NaCl, pH 7.4 |
| Acidic Elution Buffer | 0.1 M glycine, pH 2.0-3.0 |
| Neutralization Buffer | 1 M Tris-HCl, pH 8.0 |
| Storage Buffer | PBS containing 0.02% (w/v) NaN3 |
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-YFP 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-YFP 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-YFP 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-YFP 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.
Storage
4℃, 2 years
Do not dry or freeze
Components
| Components | HY-K0235-1 mL | HY-K0235-5 mL | HY-K0235-10 mL |
|---|---|---|---|
| Anti-YFP Affinity Gel | 1 mL | 5 mL | 10 mL |
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