Cat. No.: HY-K0230

Protein A/G Agarose

Name: Protein A/G Agarose
Brand: MedChemExpress
SKU: HY-K0230
Price: 271.00 USD
Availability: InStock
Rating: 5 (36 reviews)

36 Cited Publications

Customer Review

Based on 36 publication(s) in Google Scholar

MCE Protein A/G Agarose is an affinity chromatography medium for separation and purification of immunoglobulins.

Storage :
4℃, 2 years

Do not dry or freeze

Size

Price

Stock

Quantity

Amount: USD 0.00

* Please select Quantity before adding items.

Top Publications Citing

Use of MCE Products

Fluorescent Dye

Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Description & Advantages

MCE Protein A/G Agarose is an affinity chromatography medium for separation and purification of immunoglobulins.

MCE Protein A/G Agarose consists of purified Protein A/G recombinant fusion protein that has been covalently immobilized onto high-quality cross-linked 4% beaded agarose, binds nearly all isotypes and mammalian species of IgG from serum, ascites fluid, cell culture supernatant and other ant.

The product contains 0.5 mL of gel per 1 mL of total volume. Prior to use, ensure the gel is thoroughly resuspended and mixed before aspiration.

The specifications of the product correspond to the actual resin volume, with the resin content of 50%.

Protocol

Buffer Preparation

Buffer	Composition
Binding/Washing 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 8.0
Storage Buffer	1× PBS, 20% ethanol

Note: It is recommended to prepare all buffers with ultrapure water. After preparation, filter them through a 0.45 μm or 0.22 μm membrane for sterilization.

Protein Purification

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

Medium-Pressure Chromatography Column Method

1. Column Packing: Pack Protein A/G Agarose into a suitable chromatography column and connect the column to the chromatography system.

2. Column Equilibration: Equilibrate the column with 5 column volumes of Binding/Washing Buffer. Repeat 2-3 times.

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

Notes: a. Select an appropriate amount of gel according to the amount of protein. Do not exceed the binding capacity of the column;
b. Increased sample viscosity or volume may cause increased back pressure in the chromatography column.

4. Washing: Wash the column with 10-20 column volumes of Binding/Washing Buffer to remove nonspecifically adsorbed contaminating proteins. Collect the wash fractions until the UV absorbance baseline becomes stable.

5. Elution: Elute with 3-5 column volumes of Elution Buffer. Collect the eluate in separate tubes and immediately neutralize the pH with Neutralization Buffer at 1/10 of the total eluate volume. The sample may be used for subsequent functional analysis.

Note: The eluted protein may be stored at 4°C for a short period. For long-term storage, store it at -20°C.

6. Regeneration: Continue washing the column with 5-10 column volumes of Elution Buffer to completely elute the column and regenerate the resin. Then equilibrate the column to neutral pH using Binding/Washing Buffer.

Note: The agarose should be equilibrated with Binding/Washing Buffer immediately after acidic elution, and Protein A/G Agarose should not be kept in the Elution Buffer for more than 20 min.

7. Storage: Equilibrate the column with 5-10 column volumes of Storage Buffer, disconnect the column, and store it at 2-8°C.

Gravity-Flow Column Method

1. Column Packing: According to the amount of sample to be purified, transfer an appropriate volume of Protein A/G Agarose suspension into a gravity-flow chromatography column and remove the storage solution.

2. Column Equilibration: Equilibrate the packed gravity-flow column with 5 column volumes of Binding/Washing Buffer. Repeat 2-3 times.

3. Sample Loading: After adding the sample, allow it to remain in the column for at least 2 min to ensure sufficient contact between the sample and the resin. Collect the flow-through.

Note: Repeated sample loading may improve binding efficiency.

4. Washing: Wash the column with 10-15 column volumes of Binding/Washing Buffer to remove nonspecifically adsorbed contaminating proteins. Collect the wash fractions.

Note: The eluted protein may be stored at 4°C for a short period. For long-term storage, store it at -20°C.

7. Storage: Equilibrate the column with 5 column volumes of Storage Buffer and store it at 2-8°C.

Centrifugation Method

1. Agarose Pretreatment: According to the amount of sample to be purified, transfer an appropriate volume of Protein A/G Agarose suspension into a centrifuge tube. Centrifuge at 1,000 rpm for 1 min and discard the supernatant. Add 5 gel volumes of Binding/Washing Buffer, centrifuge at 1,000 rpm for 1 min, and discard the supernatant. Repeat 2-3 times.

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

3. Washing: After incubation, centrifuge at 1,000 rpm for 1 min and discard the supernatant. The supernatant may be retained as the flow-through fraction for electrophoretic analysis. Wash with 5 gel volumes of Binding/Washing Buffer, centrifuge at 1,000 rpm for 1 min, and discard the supernatant. Repeat 3-5 times.

4. Elution: Elute with 3-5 gel volumes of Elution Buffer and incubate at room temperature for 5-10 min. Centrifuge at 1,000 rpm for 1 min, collect the supernatant in separate tubes, and immediately neutralize the pH with Neutralization Buffer at 1/10 of the total eluate volume. The sample may be used for subsequent functional analysis. The elution step may be repeated 2-3 times, with each supernatant collected separately.

Note: The agarose should be equilibrated with Binding/Washing Buffer immediately after acidic elution, and Protein A/G Agarose should not be kept in the Elution Buffer for more than 20 min. The eluted protein may be stored at 4°C for a short period. For long-term storage, store it at -20°C.

5. Regeneration and Storage: Wash the resin with 5-10 gel volumes of Binding/Washing Buffer, followed by 5-10 gel volumes of ddH₂O. Finally, wash with 2 gel volumes of Storage Buffer and store at 2-8°C.

Immunoprecipitation

Preparation of Agarose

1) Add the required amount of Protein A/G Agarose suspension to a 2 mL tube, centrifuge at 1,000 rpm for 1 min and discard the supernatant.

2) Add 500 μL of Binding Buffer and mix well, centrifuge at 1,000 rpm for 1 min and discard the supernatant. Repeat 3-4 times.

Antibody Binding

1) Introduce an appropriate volume of diluted antibody into the agarose, gently rotate the tube for 30 min at room temperature or 2 h at 4°C. Centrifuge at 1,000 rpm for 1 min and retain the supernatant for future analysis.

2) Add 500 μL Washing Buffer to the agarose and mix well, rotate the tube for 3-5 min at room temperature to eliminate hetero-proteins that have been non-specifically adsorbed. Centrifuge at 1,000 rpm for 1 min and discard the supernatant. Repeat 2-3 times.

Immunoprecipitation of Target Antigen

1) Add the antigen-containing sample to the agarose and mix well, then rotate the tube for 30 min at room temperature or leave overnight at 4°C.

2) Centrifuge at 1,000 rpm for 1 min and retain the supernatant for future analysis.

3) Add 1 mL Washing Buffer to the agarose and mix thoroughly, centrifuge at 1,000 rpm for 1 min and discard the supernatant. Repeat the washing procedure until the OD₂₈₀ of the supernatant is below 0.05.

Note: If the OD₂₈₀ of the supernatant is greater than 0.05, increase the number of washing steps as appropriate.

4) Add 1 mL Washing Buffer to the agarose again, mix well and transfer to a new tube.

Antigen Elution

Two elution methods are provided. Select the appropriate method according to the downstream analysis.

1) Acidic Elution: Add 50-100 μL of Elution Buffer, mix thoroughly and incubate at room temperature for 5-10 min. Centrifuge at 1,000 rpm for 1 min, collect the supernatant and immediately neutralize the pH with Neutralization Buffer at 1/10 of the total eluate volume.

2) Denaturing Elution: Add 25 μL of 1× SDS-PAGE Loading Buffer, mix thoroughly and heat at 95°C for 5 min. Centrifuge at 1,000 rpm for 1 min and collect the supernatant for SDS-PAGE analysis.