MagVigen™ – anti-His-Tag nanoparticles are ideal for His-Tag protein purification and immunoprecipitation assays. MagVigen™ anti-His-Tag recognizes and efficiently binds to His-Tag molecules following a short incubation. The generated nanoparticle-His-Tag molecule complex can be separated from the rest of the sample by magnet. The retained antibody can be eluted from the nanoparticles using an elution buffer.
MagVigen™ nanoparticles enable identification of new protein-protein interactions through immunoprecipitation assays, where the MagVigen™ anti-His-Tag – His-Tag molecule complex can be used to isolate particular proteins of interest or protein complex from assay samples, e.g. cell lysate. The immunoprecipitated proteins can be further analyzed by electrophoresis, protein staining, and mass spectrometry.
MagVigen™ nanoparticles are much smaller than conventional micro-beads. This feature allows for better accessibility of the nanoparticles to the antigenic epitope and for less disturbance to the native functions of proteins or protein-protein complexes. In addition, the surfaces of MagVigen™ nanoparticles are uniquely coated to reduce non-specific interactions with cellular proteins and other biomolecules. This feature allows for a more specific “pull down” of real protein complex targets.
- Cat# 51009: MagVigen™ – Anti-FLAG nanoparticles (Cat# 51007) are provided in phosphate buffered saline (PBS), pH 7.4. Each vial contains 1 ml of solution with a particle concentration of 1 mg/ml, which is enough for approximately 20-200 antibody enrichment or immunoprecipitation assays.
Cat# K51009 further includes:
- Washing Buffer (10X), 15 ml
All materials should be stored at 4°C.
Shelf life: 6 months
This protocol was optimized for enrichment of 1-10 μg mouse antibody in a volume of 100 μl . For a smaller size of sample, it is recommended to add extra Washing Buffer to reach a 100 μl reaction volume. For larger scale of purification, adjust the amount of reagents accordingly.
- Dilute 10X Washing Buffer with PBS to 1X.
- Vortex MagVigen™ nanoparticles for 10-20 seconds.
- Take 5-50 μl nanoparticle solution (for 1-10 μg antibody), add it to 100 μl 1X Washing Buffer, and vortex to mix.
- Separate the nanoparticles from the solution by placing the magnet on the side of the tube for 2-5 min and remove the supernatant carefully (with magnet still on the side).
- Remove magnet and wash the nanoparticles with 100 μl 1X Washing Buffer. Repeat step 4, and remove supernatant.
- Add 100 μl sample solution containing desired antibodies to the nanoparticle pellet, mix well, and incubate with gentle rotation for 2 hours at room temperature or 4 °C overnight.
- After incubation, use the magnet to separate nanoparticle-antibody complex from the solution and remove the supernatant.
- Wash nanoparticle-antibody complex with 100 μl 1X Washing Buffer twice and remove supernatant.
- Elute captured antibody from the nanoparticles by adding 90 μl Elution Buffer (customer choice), mix well, and incubate for 1 min at room temperature.
- Separate the nanoparticles from the eluted antibody with magnet. Transfer supernatant to a clean tube and immediately neutralize the eluate by adding 10 μl Tris (1M, pH=8.0). The enriched antibody is ready to use for subsequent evaluation.
Note: A clear precipitate containing dark brown colored nanoparticles should become visible on the side of the micro-centrifuge tube.
Immunoprecipitation (from cell lysate)
Steps 1-8 are the same as Antibody Enrichment
- Add cell lysate sample, typically 100-1000 μl , to nanoparticle pellet and gently pipette to mix.
- Incubate the reaction by rotating for 1-2 hours at room temperature or 4°C to allow the antigen to bind to the MagVigen™-antibody complex.
- After incubation, use the magnet to separate the nanoparticle-antibody-protein complex from the solution, and remove the supernatant.
- Wash nanoparticle-antibody-protein complex with 100 μl of 1X Washing Buffer for three times.
- Elute antibody and proteins by using either the denaturing elution methods or the non-denaturing elution method.
Note: depending on the affinity of the antibody used, the incubation time can be adjusted for optimal binding.
A. Denaturing elution:
1) Add 20-30 μl of SDS-PAGE protein sample buffer to the nanoparticle-antibody-protein complex, gently pipette, and boil the sample in water bath for 5 minutes.
2) Place the tube on the magnet to separate the nanoparticles, and load the supernatant onto a gel.
B. Non-denaturing elution:
1) Add 20-30 μl of Elution Buffer to the nanoparticle-antibody-protein complex, gently pipette, and incubate for 1-2 minutes.
2) Place the tube on the magnet to separate the nanoparticles, and transfer the supernatant to a clean tube. If neural pH is desired for further analysis, add Tris (1 M, pH=8) to the sample.