His Tag Immunoprecipitation Kit,IC-8122

Product Description  

His Tag immunoprecipitation magnetic beads specifically bind to His-tagged proteins and can be used for immunoprecipitation (IP) of proteins, protein complexes, protein-nucleic acid complexes, and other antigens. The kit includes optimized pre-formulated buffers to provide the best reaction conditions for immunoprecipitation experiments, enhancing experimental stability and reproducibility. It is suitable for antigen samples from cell lysates, cell culture supernatants, serum, ascites, and other sources.  

Product Features  

1. Low non-specific adsorption.  

2. High efficiency, high yield, low consumption.  

3. Simple and flexible operation.  

4. Reliable and reproducible results.  

Kit Components  

- His Tag Immunomagnetic Beads: 1 mL  

- Cell Lysis Buffer: 20 mL  

- Washing Buffer (10¡Á): 20 mL  

- Acidic Elution Buffer: 4 mL  

- Neutralization Buffer: 1 mL  

Operating Procedure  

1. Preparation of Cell Lysate  

Treat cell samples with Cell Lysis Buffer and prepare the cell lysate according to standard protocols. Keep on ice for immediate use or store at -20¡æ for long-term storage.  

2. Magnetic Bead Pretreatment  

2.1 Vortex the immunomagnetic beads for 1 minute to resuspend them. Transfer 25 µL of the bead suspension into a 1.5 mL EP tube.  

2.2 Dilute the Washing Buffer (10¡Á) to 1¡Á with distilled water. Add 500 µL of Washing Buffer (1¡Á) to the EP tube and gently invert the tube several times to resuspend the beads. Place the EP tube in a magnetic separator for 1 minute, then discard the supernatant. Repeat the washing step twice.  

3. Immunoprecipitation  

3.1 Add 500 µL of the prepared cell lysate to the EP tube. Place the tube on a rotator and mix at 37¡æ for 30 minutes. For weaker binding, incubate at room temperature for 1 hour or at 4¡æ overnight.  

3.2 After incubation, perform magnetic separation and discard or save the supernatant for further analysis.  

3.3 Add 500 µL of Washing Buffer to wash the beads. Perform magnetic separation, discard the supernatant, and repeat the washing step three times.  

4. Antigen Elution  

4.1 Denaturing Elution: Add 100 µL of SDS-PAGE Loading Buffer (self-prepared), mix well, and heat at 95¡æ for 5 minutes. Perform magnetic separation or centrifuge (13,000g, 10 minutes, room temperature), then collect the supernatant for SDS-PAGE analysis.  

4.2 Neutral Elution: Add 50 µL of His Peptide Elution Buffer (PBS, 1 mg/mL 6¡ÁHis peptide, pH 7.4) to the EP tube. Incubate on a rotator at 37¡æ for 5-10 minutes (extend incubation time if below 37¡æ). Perform magnetic separation or centrifugation and collect the supernatant. Repeat the step to improve antigen recovery.  

4.3 Acidic Elution: Add 100 µL of Acidic Elution Buffer to the EP tube. Incubate on a rotator at 37¡æfor 5-10 minutes. Perform magnetic separation or centrifugation and collect the supernatant. To neutralize the acidic eluate, add 50 µL of Neutralization Buffer to 100 µL of eluate and adjust the pH to neutral.  

Storage Conditions  

Lysis Buffer: -20¡æ, 2 years. Other Reagents: 4¡æ, 2 years.  

Precautions  

1. Avoid freezing the magnetic beads. Store the beads in the provided storage solution to prevent drying.  

2. Magnetic separation should last at least 1 minute to minimize bead loss.  

3. Vortex the beads thoroughly before use to ensure even suspension. Avoid generating bubbles during operation.  

4. Use high-quality pipette tips and reaction tubes to prevent loss due to bead or solution adhesion.  

5. Supernatants from antibody or antigen binding steps can be used to assess the binding efficiency of antibodies, antigens, and beads.  

6. If the provided buffer system does not yield ideal results, users can screen and prepare their own buffers.  

7. This product is for research use only and should not be used for clinical diagnosis, treatment, food, or drugs. Do not store in residential areas.  

8. Wear a lab coat and disposable gloves for safety and health protection during operation.  

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