Anti-V5 Magnetic Beads

Antigen Sample Preparation

Serum: If the target protein abundance is high, it is recommended to dilute the serum sample to a final target protein concentration of 50-100 μg/mL and keep on ice (or store at -20°C for long-term storage).

Suspended Cells: 1. Centrifuge to collect cells (4°C, 500 g, 10 min), discard the supernatant, weigh the pellet and wash with PBS (1×) twice. 2. Add lysis buffer at a ratio of 5-10 μL/mg of cells, along with an appropriate amount of protease inhibitor. Mix well and incubate on ice for 10 min for complete lysis. 3. Centrifuge (4°C, 14,000 g, 10 min) and collect the supernatant. Store on ice or at -20°C for long-term storage.

Adherent Cells: 1. Carefully remove culture medium from confluent cells and wash the cells twice with PBS (1×). 2. Scrape off the cells with a cell scraper and collect them in a 1.5 mL EP tube. Add lysis buffer at a ratio of 20-30 μL/1×10⁵ cells, along with an appropriate amount of protease inhibitor. Mix well and incubate on ice for 10 min for complete lysis. 3. Centrifuge (4°C, 14,000 g, 10 min) and collect the supernatant. Store on ice or at -20°C for long-term storage.

Note: It is recommended to choose an appropriate lysis buffer based on the sample type. IP Lysis Buffer (MCE Cat. No. HY-K1000) is highly recommended.

Preparation of Magnetic Beads

Fully resuspend the magnetic beads and transfer the desired volume into a 1.5 mL EP tube. Add 500 μL of wash buffer, mix thoroughly, place the tube on a magnetic rack to separate, and discard the supernatant. Repeat the washing step two more times.

Note: a. To ensure uniform bead distribution, it is recommended to mix the beads thoroughly by repeated inversion, gentle vortexing, or using a mixer.

b. Use 20 μL of magnetic beads for every 500 μL of cell lysate.

IP

1. Binding: Add the sample containing the target protein to the pretreated magnetic beads. Incubate the mixture on a rotator at 4°C for 2-4 h or at room temperature for 1-2 h (incubation time can be adjusted based on binding efficiency).

2. Washing: After incubation, perform magnetic separation and collect the supernatant (optional: save the supernatant for SDS-PAGE analysis). Wash the beads with 1 mL of wash buffer, mix, and separate magnetically. Discard the supernatant. Repeat this washing step 2-3 times to obtain the bead-protein complex.

3. Elution: Two elution methods are provided. It is recommended to choose the appropriate method based on downstream applications.

1) Acidic Elution: This method preserves the biological activity of the eluted protein, suitable for downstream functional assays.

Add 100 μL of elution buffer to the beads, mix thoroughly, and incubate at room temperature for 5-10 min. Perform magnetic separation and immediately neutralize the eluted supernatant with neutralization buffer (1/10 of the elution volume).

Note: The eluted protein can be stored at 4°C for short-term use or at -20°C for long-term storage.

2) Denaturing Elution: This method is suitable for SDS-PAGE analysis.

Add 100 μL of protein loading buffer (1×) to the beads, mix thoroughly, and heat at 95°C for 10 minutes. Perform magnetic separation and collect the supernatant for electrophoresis.