Nanoselector Agarose Immunoprecipitation Protocol

Introduction

 

HUABIO’s Nanoselector technology is composed of our recombinant high-affinity Nanobodies covalently coupled to agarose beads. Our cutting-edge nanobodies are camelid antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain. They are the smallest functional antigen-binding fragment and do not contain light chains. The recombinant high-affinity nanobodies eliminate the batch-to-batch variations.

 

Nanoselector IP Protocol

Recipes for all referenced solutions can be found here.

 

Sample Preparation

Cell harvest and cell lysis should be performed with ice-cold Lysis Buffers. To prevent protein degradation, add protease inhibitors to the Lysis Buffer. For one immunoprecipitation reaction, we recommend using approximately 10^6 - 10^7 cells.

  • For cytoplasmic proteins, resuspend the cell pellet in 200 μL ice-cold Lysis Buffer by pipetting up and down.

  • For nuclear/chromatin proteins, resuspend the cell pellet in 200 μL ice-cold RIPA Buffer.

  • Incubate on ice for 30 minutes. Disrupt the cells every ten minutes with repeated agitation.

  • Centrifuge the cell lysate at 17,000xg for 10 minutes at +4°C. Transfer supernatant to a pre-cooled tube and add 300 μL Dilution Buffer. If necessary, set aside 50uL diluted lysate (input fraction) for later analysis.

Bead Equilibration

  • Gently resuspend the beads by pipetting up and down or by inverting the tube. Do not vortex.

  • Transfer 25 μL of bead slurry into a 1.5 mL reaction tube.

  • Centrifuge at 2,500xg for 5 minutes at +4°C.

  • Discard the supernatant.

Protein Binding

  • Add diluted cell lysate to the equilibrated beads.

  • Rotate end-over-end for 1 hour at +4°C.

  • Centrifuge at 1,000xg for 5 minutes at 4° C. If necessary, set aside 50uL diluted lysate for further analysis.

  • Carefully aspirate and discard the supernatant.

  • Resuspend beads in 500 μL Wash buffer.

  • Centrifuge at 1,000 xg for 5 minutes at +4° C

  • Carefully aspirate and discard the supernatant.

  • Resuspend beads in 500 μL Wash buffer. Repeat centrifugation and wash step twice more.

  • During the last resuspension, transfer the beads to a new tube.

Elution with 2x SDS-Sample Buffer

  • Carefully aspirate and discard the supernatant.

  • Resuspend beads in 80 μL 2x SDS-sample buffer.

  • Boil for 5 minutes at+95°C to dissociate immunocomplexes from beads.

  • Centrifugate at 2,500x g for 2 min at +4°C.

  • Analyze the supernatant via SDS-PAGE.

Only for research applications, not for diagnostic or therapeutic use.

 

View: Suggested Buffers

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