Anti-Flag Affinity Gel

General Protocol

1. Preparation of Anti-Flag Affinity Gel

1.1 Thoroughly suspend the Anti-Flag Affinity Gel. Transfer 10 μL of the gel suspension (about 5 μL of settled gel) to a clean tube.

1.2 Add 600 μL of Wash Buffer to resuspend the gel. Centrifuge at 10,000 rpm for 30 seconds. Remove the supernatant carefully. Be sure that most of the wash buffer is removed and no gel is discarded. Repeat 3-4 times.

2. Protein Binding

2.1 Add 500 μL of cell lysate (the sample containing Flag-tagged protein) to the cleaned gel from step 1.2. Incubate for 2 hours at 4°C while gently rotating the tube. For higher binding efficiency, please incubate overnight.

2.2 Centrifuge at 10,000 rpm for 30 seconds, and transfer the supernatant to a new cube (the supernatant can be used to determine whether there is residual Flag-tagged protein).

3. Washing

Wash the gel with 500 μL of Wash Buffer, centrifuge at 10,000 rpm for 30 seconds. Remove the supernatant carefully. Be sure that most of the wash buffer is removed and no gel is discarded. Wash until the OD280 of the supernatant liquid＜0.05.

4. Elution/Detection

Three elution methods are recommended according to protein characteristics or further usage:

4.1 Elution with SDS-PAGE Loading Buffer for gel electrophoresis and immuoblotting.

Add 50 μL of 1× SDS-PAGE Loading Buffer to each tube. Mix well and boil for 5 minutes. Centrifuge at 10,000 rpm for 30 seconds. Keep the supernatant for SDS-PAGE analysis.

4.2 Elution with Elution Buffer A under acidic condition.

Add 50 μL of Elution Buffer A to each tube. Mix well and incubate for 10 minutes at room temperature. Centrifuge at 10,000 rpm for 30 seconds and transfer the supernatant to a new tube. Add 25 μL of Neutralization Buffer for each 50 μL of eluate to neutralize the low pH, which may help preserve bioactivity of target proteins.

4.3 Elution with Elution Buffer B under native condition.

Add 30-50 μL of Elution Buffer B to each tube. Incubate with gentle shaking or on a rotator for 1 hour at room temperature or 2 hours at 4°C. Centrifuge at 10,000 rpm for 30 seconds and transfer the supernatant to a new tube.

NOTE: For immediate use, store the eluates at 4°C, or store at -20°C for long term storage.

Precautions

1. Please resuspend the gel thoroughly before use.

2. For the best results, determine optimal conditions for expression of Flag-tagged fusion protein before attempting immunoprecipitation.

3. To minimize protein degradation, protease inhibitor cocktails are highly recommended.

4. Do not use cell lysate containing dithiothreitol (DTT). DTT may cause the Anti-Flag antibody to leach from the gel.

5. This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.