· Consistent and reproducible results
· No heavy & light antibody chains
· Extraordinary binding, even under harsh conditions
· High affinity
· Short Incubation (5-30 min)
Description:
RFP Nanoselector Magnetic beads have been specifically designed to bind RFP-fusion proteins. RFP Nanoselector Magnetic beads is based on small high-affinity recombinant alpaca antibody fragments covalently coupled to the surface of Magnetic beads. RFP Nanoselector Magnetic beads are ideal tools to isolate or purify RFP-fusion proteins fast and efficiently.
Ligand: Anti-RFP single domain antibody fragment (VHH, Nanobody)
Bead size: ~ 2.8µm
Reactivity: Recognizes mCherry, mRFP, mRFPruby, mPlum, tagRFP, mKate2 and many more RFP derivatives.
Binding capacity: High binding capacity, 10 µL slurry bind about 20 µg of recombinant RFP.
Storage: Shipped at ambient temperature. Upon receipt store at 4°C. Stable for 1 year. Do not freeze.
Storage Buffer: 50 % slurry in PBS containing 20 % Ethanol
Background:
Red fluorescent proteins and the variants thereof are widely used to study protein localization and dynamics. For biochemical analysis including mass spectrometry and enzyme activity measurements these RFP-fusion proteins and their interacting factors need to be isolated fast and efficiently by immunoprecipitation using the RFP Nanoselector Magnetic beads. Due to the single-chain nature of sdAbs and their stable and covalent attachment, no leakage of light and heavy chains is observed during elution with SDS sample buffer.
Immunoprecipitation (IP)/Co-IP
Mass spectrometry (MS)
Enzyme activity measurements
Immunoprecipitation protocol
Mammalian cell lysis
Note: Harvesting of cells and cell lysis should be performed with ice-cold buffers. We strongly recommend to add protease inhibitors to the Lysis buffer to prevent degradation of your target protein and its binding partners.
For one immunoprecipitation reaction, we recommend using ~106- 107 cells.
1. Choice of lysis buffer:
* For cytoplasmic proteins, resuspend the cell pellet in 200 µL ice-cold Lysis buffer by pipetting up and down. Supplement Lysis buffer with protease inhibitor cocktail and 1 mM PMSF (not included).
* For nuclear/chromatin proteins, resuspend cell pellet in 200 µL ice-cold RIPA buffer supplemented with DNaseI (f.c. 75-150 Kunitz U/mL), MgCl2 (f.c. 2.5 mM), protease inhibitor cocktail and PMSF(f.c. 1 mM)(not included)
2. Place the tube on ice for 30 min and extensively pipette the suspension every 10 min.
3. Centrifuge cell lysate at 17,000x g for 10 min at +4°C. Transfer cleared lysate (supernatant) to a pre cooled tube and add 300 µL Dilution buffer supplemented with 1 mM PMSF and protease inhibitor cocktail (not included). If required, save 50 µL of diluted lysate for further analysis (input fraction).
Beads equilibration
1. Resuspend the beads by gently pipetting up and down or by inverting the tube. Do not vortex the beads!
2. Transfer 25 µL of bead slurry into a 1.5 mL reaction tube.
3. Add 500 µL ice-cold Dilution buffer.
4. Separate the beads with a magnet until the supernatant is clear.
5. Discard the supernatant.
Protein binding
1. Add diluted lysate to the equilibrated beads.
2. Rotate end-over-end for 1 hour at +4°C.
Washing
1. Separate the beads with a magnet until the supernatant is clear.
2. If required, save 50 µL of supernatant for further analysis(flow-through/non-bound fraction).
3. Discard remaining supernatant.
4. Resuspend beads in 500 µL Wash buffer.
5. Separate the beads with a magnet until the supernatant is clear. Discard the remaining supernatant.
6. Repeat this step at least twice.
7. During the last washing step, transfer the beads to a new tube.
Optional: To increase stringency of the Wash buffer, test various salt concentrations e.g. 150 mM - 500 mM,and/or add a non-ionic detergent e.g. Triton™ X-100.
Elution with 2x SDS-sample buffer
1. Remove the remaining supernatant.
2. Resuspend beads in 80 µL 2x SDS-sample buffer.
3. Boil beads for 5 min at +95°C to dissociate immunocomplexes from beads.
4. Separate the beads with a magnet.
5. Analyze the supernatant in SDS-PAGE.
Elution with Glycine-elution buffer
1. Remove the remaining supernatant.
2. Add 50–100 µL Glycine-elution buffer and constantly pipette up and down for 30 - 60 sec at +4°C.
3. Separate the beads with a magnet until the supernatant is clear..
4. Transfer the supernatant to a new tube.
5. Immediately neutralize the eluate fraction with Neutralization buffer.
6. Repeat this step at least once to increase elution efficiency .
