RFP-Trap® MA

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Antigen
Reactivity
Discosoma
6
1
1
Host
Camelid (Camelidae)
Antibody Type
Recombinant Antibody
Conjugate
Magnetic Agarose Beads
Application
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Mass Spectrometry (MS), Immunoprecipitation (IP), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
Options
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Purpose RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (magnetic agarose beads) for biochemical analysis of RFP fusion proteins and their interacting partners.
Brand RFP-Trap®
Sample Type Cell Extracts
Specificity Binding capacity: 10 μL RFP-Trap®_MA slurry binds 3 - 4 μg of RFP
Cross-Reactivity (Details) tested on RFP, mCherry, mOrange, mPlum, tagRFP
Characteristics Antibodies - extremely powerful tools in biomedical research - are large complex molecules (~ 150 kDa) consisting of two heavy and two light chains. Due to their complex structure, the use of antibodies is often limited and hindered by batch-to-batch variations.

Camelidae (camels, dromedaries, llamas and alpacas) possess functional antibodies devoid of light chains, so-called heavy chain antibodies (hcAbs). hcAbs recognize and bind their antigens via a single variable domain (VHH). These VHH domains are the smallest intact antigen binding fragments (~ 13 kDa).

Nano-Traps are based on single domain antibody fragments (VHHs) derived from alpaca.
Components RFP-Trap® coupled to magnetic agarose beads
Material not included Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
Alternative Name RFP
Background RFP fluorescent proteins (RFPs) and variants thereof are widely used to study proteinlocalization and dynamics. For biochemical analysis including mass spectrometry and enzyme activity measurements these RFP-fusion proteins and their interacting factors can be isolated fast and efficiently by immunoprecipitation using the RFP-Trap®. RFP-Trap® utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
Research Area Tags/Labels
Application Notes Optimal working dilution should be determined by the investigator.
Comment

Particle size ~ 40 μm

Assay Time 1.5 h
Protocol - Robust and versatile tool for biochemical analyses of RFP-fusion proteins
- Short incubation times (5 - 30 min)
- Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
- Low unspecific binding
- No contaminating heavy and light chains of conventional antibodies
- Applicable in Chromatin Immunoprecipitation (ChIP)
Reagent Preparation

Suggested buffer composition

- Lysis buffer (CoIP): 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA,0.5 % NP-40
- 10x RIPA buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1 % SDS, 1 % Triton X-100, 1 % Deoxycholate
- Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
- Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
- Elution buffer: 200 mM glycine pH 2.6

Sample Collection Harvest cells:
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) expressing a RFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 mLice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4 °C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.

Lyse cells
1. Resuspend cell pellet in 200 μL ice-cold lysis buffer by pipetting or using a syringe.
note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included). optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).

2. Place the tube on ice for 30 min with extensively pipetting every 10 min.

3. Centrifuge cell lysate at 20.000x g for 10 min at +4 °C. Transfer lysate to a pre-cooled tube. Add 300 μL dilution buffer to lysate. Discard pellet.
note: At this point cell lysate may be put at -80 °C for long-term storage. optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer
We recommend that during incubation with the beads the final concentration of detergents does not exceed 0.2 % to avoid unspecific binding to the matrix. If required, use more dilution buffer to dilute the supernatant accordingly.
Assay Procedure

Equilibrate beads
4. Vortex RFP-Trap®_MA beads and pipette 25 μL bead slurry into 500 μL ice-cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
Bind proteins:
5. Add diluted lysate (step 3) to equilibrated RFP-Trap®_MA beads (step 4). If required, save 50 μL of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4 °C.
6. Magnetically separate beads until supernatant is clear. If required, save 50 μL supernatant for immunoblot analysis. Discard remaining supernatant.
Wash beads:
7. Resuspend RFP-Trap®_MA beads in 500 μL dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
optional: Increase salt concentration in the second washing step up to 500 mM.
Elute proteins:
8. Resuspend RFP-Trap®_MA beads in 100 μL 2x SDS-sample buffer.
9. Boil resuspended RFP-Trap®_MA beads for 10 min at 95 °C to dissociate immunocomplexes from RFP-Trap®_MA beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant.
10. optional instead of steps 8 and 9: elute bound proteins by adding 50 μL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by magnetic separation. Transfer the supernatant to a new tube and add 5 μL 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.

Restrictions For Research Use only
Buffer Storage buffer: 20 % EtOH
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
 image for RFP-Trap® MA (ABIN2452223) RFP-Trap® MA
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