Protein G Kit

• Cat.No.: Kit-0888
• Product Overview: Protein G Kit includes silica based magnetic nanobeads conjugated with highly purified Protein G (Purity >95%) and buffers required for protein purification. Protein G Kit allows simple and quick experiment due to a fast magnetic response rate. Protein G Kit is not only for antibody purification, but for other immunoprecipitation purpose, such as antigen purification, proteinprotein interaction, and cell separation.

Specification

• Tag: Non
• Storage: Protein G Magnetic NanoBeads are supplied as a 4% (w/v) suspension in storage buffer and should be stored at 2~8°C.
• Kit Components: Protein G Magnetic NanoBeads 1 mL X 1 ea(4%);
  Binding & Washing buffer 20 mL x 2 ea;
  Elution Buffer 1 mL X 1 ea;
  Neutralization Buffer 1 mL X 1 ea;
  Manual 1 ea.
  Protein G Magnetic NanoBeads contain 40 mg Beads/mL in storage buffer (phosphate buffered saline, pH 7.4, 0.02% Tween-20, and 0.1% NaN3).
• Features & Benefits: Fast Binding: Powerful magnetism reduces experiment time and increases yield.
  Large Surface Area: Average diameter of 400nm provides large surface and allows high binding capacity.
  Specificity: Globular beads reduce non-specific binding.
• Assay Protocol: 1. Immunoglobulin purification
  Preparation of Protein G Magnetic Nanobeads
  1. Resuspend Protein G Magnetic NanoBeads by gently vortexing the tube.
  2. Transfer 200 μL of Protein G Magnetic NanoBeads into a 1.5 mL tube.
  3. Place the tube on a magnet to pull down the beads and remove supernatant.
  4. Add 1 mL of Binding & Washing buffer and wash the beads.
  5. Place the tube on a magnet and remove supernatant.
  Binding Immunoglobulin
  1. Add 500 μL of sample, and 500 μL of Binding & Washing buffer to the tube containing the beads. Incubate in a rotator for 1 hour at room temperature.
  Note) Make sure that the beads are resuspended well. This is important for efficient purification.
  2. Place the tube on a magnet and collect supernatant.
  Note) Keep supernatant for SDS-PAGE to check binding.
  3. Add 500 μL of Binding & Washing buffer to the tube and mix gently to wash the beads. Place the tube on a magnet and remove supernatant. Repeat this process two times more.
  Elution
  1. Add 100 μL of Elution buffer to the tube and mix well by pipetting. Incubate for 1 minute.
  2. Place the tube on a magnet and transfer elute to a new tube. Repeat this process one more.
  Note) For better yield, repeat the elution step one more or increase elution buffer volume.
  3. Add 10 μL (10% of elute) of Neutralization buffer.
  
  2. Immunoprecipitation
  This protocol offers a general guideline for immunoprecipitation. Optimization might be required for your research.
  Preparation of Protein G Manetic Nanobeads
  1. Resuspend AccuNanoBead™ Protein G Magnetic NanoBeads by gently vortexing the tube.
  2. Transfer 50 μL of AccuNanoBead™ Protein G Magnetic Nanobeads into a 1.5 mL tube.
  3. Place the tube on a magnet and remove supernatant.
  4. Add 250 μL of Binding & Washing buffer and wash the beads.
  5. Place the tube on a magnet and remove supernatant.
  Binding Antibody & Antigen
  1. Add 1~10 μg antibody in final volume of 200 μL solution to the tube containing the beads and resuspend it well.
  2. Incubate in a rotator for 10 minutes at room temperature.
  3. Place the tube on a magnet and remove supernatant.
  4. Add 250 μL of Binding & Washing buffer to the tube and wash the beads. Place the tube on a magnet to remove supernatant. Repeat this process two times more.
  5. Add reagent containing antigen (100~1,000 μL) and mix with beads well.
  6. Incubate in a rotator for 1 hour at room temperature.
  Note) Resuspend the beads well.
  7. Place the tube on a magnet and collect supernatant.
  Note) Keep the supernatant for SDS-PAGE to check binding.
  8. Add 200 μL of Binding & Washing buffer to the tube containing the beads and mix gently to wash the beads. Place the tube on a magnet and remove supernatant.
  Repeat this process two times more.
  9. Add 200 μL of Binding & Washing buffer and resuspend well. Transfer it to a new tube.
  10. Centrifuge the tube containing beads at 1,000~3,000 rpm for 1~3 sec, briefly.
  11. Place the tube on a magnet and remove Binding & Washing buffer as much as possible.
  Elution
  A. Denaturing elution
  1. Add 20 μL of Elution buffer to the tube and mix well by pipetting.
  2. Incubate at 70℃ for 10 minutes.
  3. Place the tube on a magnet and transfer elute to a new a tube.
  4. Add reducing agent containing SDS-PAGE sample buffer and mix well. Load the sample to SDS-PAGE.
  B. Non-denaturing elution
  1. Add 20 μL of elution buffer to the tube and mix well by pipetting.
  2. Incubate for 2 minutes. Place the tube on a magnet and transfer elute to a new tube.
  3. Add 2 μL (10% of elute) of Neutralization buffer.
  Note) User may utilize other buffer for neutralization depending on the objective of experiment.