Protein A Agarose (Alkali-Tolerant) 4FF
Based on 1 Customer Validation
MCE Protein A Agarose (Alkali-Tolerant) 4FF can efficiently binds IgG from serum, ascites, culture supernatants, and other antibody-containing samples, enabling high-performance antibody purification.
-
Storage :
4°C, 5 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
Protein A is a cell wall protein originally isolated from Staphylococcus aureus, known for its specific binding to the Fc region of mammalian immunoglobulin G (IgG) heavy chains. Native protein A contains five IgG-binding domains, along with several additional domains of unknown function. In contrast, recombinant protein A retains only the IgG-binding domains, significantly reducing nonspecific adsorption.
MCE Protein A Agarose (Alkali-Tolerant) 4FF is manufactured by covalently coupling recombinant protein A to a highly cross-linked agarose matrix. It features high binding capacity, excellent specificity, and strong ligand stability. The resin is alkali-tolerant and can withstand in-place cleaning with 0.5 M NaOH, making it suitable for repeated use. This product efficiently binds IgG from serum, ascites, culture supernatants, and other antibody-containing samples, enabling high-performance antibody purification.
Each 1 mL of total volume contains 0.5 mL of resin. Thoroughly resuspend the resin before use and mix well before aliquoting.
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 | 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0 |
| Elution Buffer | 0.1 M glycine, pH 3.0 |
| Neutralization Buffer | 1 M Tris-HCl, pH 8.5 |
| Storage Buffer | 1× PBS containing 20% ethanol |
Note: Prepare all buffers with high-purity water and sterilize them by filtration through a 0.45 μm or 0.22 μm membrane.
Sample Preparation
Before loading, centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane. Dilute or dialyze serum, ascites, or culture supernatant against binding/wash buffer to obtain a suitable ionic strength and pH.
Medium-Pressure Chromatography Column Method
1. Column Packing: Thoroughly resuspend the resin, pack it into a suitable chromatography column, and connect the column 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.
4. Washing: Wash with 10-20 column volumes of binding/wash buffer until the OD280 or UV absorbance reaches a stable baseline.
5. Elution: Elute with 3-5 column volumes of 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 elution buffer to remove residual protein, then equilibrate to neutral pH with binding/wash buffer. Limit exposure to acidic elution buffer to 20 min.
7. Cleaning-in-Place: Remove ionically bound contaminants with 5-10 column volumes of 2 M NaCl, or perform alkaline cleaning with 3-5 column volumes of 0.1-0.5 M NaOH. Immediately rinse thoroughly with deionized water and binding/wash buffer.
8. Storage: Equilibrate with 5-10 column volumes of storage buffer and store at 2-8°C.
Gravity-Flow Column Method
1. Column Packing and Equilibration: Pack the resin into a gravity-flow column, remove the storage solution, and equilibrate with 5 column volumes of binding/wash buffer 2-3 times.
2. Sample Loading: Allow the sample to remain in the column for at least 2 min, then collect the flow-through. Repeated loading may improve binding efficiency.
3. Washing: Wash with 10-15 column volumes of binding/wash buffer and collect wash fractions.
4. Elution: Elute with 3-5 column volumes of elution buffer, collect fractions, and immediately add neutralization buffer at 1/10 of the total eluate volume.
5. Regeneration and Storage: Remove residual protein with elution buffer, equilibrate to neutral pH with binding/wash buffer, then equilibrate with 5 column volumes of storage buffer and store at 2-8°C.
Centrifugation Method
1. Resin Pretreatment: Transfer an appropriate amount of resin suspension into a centrifuge tube, centrifuge at 1,000 rpm for 1 min, discard the supernatant, and wash with 5 resin 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 1,000 rpm for 1 min and wash with 5 resin volumes of binding/wash buffer 3-5 times.
4. Elution: Add 3-5 resin volumes of elution buffer, incubate at room temperature for 5-10 min, centrifuge at 1,000 rpm for 1 min, collect the supernatant, and immediately add 1/10 volume of neutralization buffer. Repeat 2-3 times if necessary.
5. Regeneration and Storage: Wash sequentially with 5-10 resin volumes of binding/wash buffer, 5-10 resin volumes of ddH2O, and 2 resin volumes of storage buffer. Store at 2-8°C.
Storage
4°C, 5 years.
Do not dry or freeze.
Attention
1. Ensure the resin is thoroughly resuspended before use.
2. It is recommended to replace the storage buffer every 2-3 months to prevent microbial contamination due to ethanol evaporation.
3. To minimize protein degradation, protease inhibitor cocktails (MCE Cat. No. HY-K0010, HY-K0011) are highly recommended.
4. This product is for R&D use only, not for drug, household, or other uses.
5. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Components | HY-K0251-1 mL | HY-K0251-5 mL | HY-K0251-25 mL | HY-K0251-100 mL |
|---|---|---|---|---|
| Protein A Agarose (Alkali-Tolerant) 4FF |
1 mL | 5 mL | 25 mL | 100 mL |
Please enter the correct Lot Number
