Myc-Trap® Agarose Kit
- Recombinant Antibody
- Camelid (Camelidae)
- Agarose Beads
- 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)
- The ChromoTek Myc-Trap® comprises an antibody fragment coupled to agarose beads for immunoprecipitation of Myc-tagged proteins.
- Cell Extracts
- The Myc-Trap® recognizes the Myc-tag sequence EQKLISEEDL at the N-terminus, C-terminus, or internal site of the fusion protein.
- Epitope tags are useful for the labelling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein's biochemical properties. The Myc epitope tag is widely used to detect expression of recombinant proteins in bacteria, yeast, insect and mammalian cell systems. For biochemical analysis including mass spectrometry and enzyme activity measurements these Myc-tagged proteins and their interacting factors can be isolated fast and efficiently by immunoprecipitation using the Myc-Trap®. Myc-Trap® utilizes small recombinant antibody fragments covalently coupled to the surface of agarose beads.
- Myc-Trap® coupled to agarose beads
- Optimal working dilution should be determined by the investigator.
Bead size ~ 90 μm (cross-linked 4% agarose beads)
- 1.5 h
- Aufbereitung der Reagenzien
4. Vortex Myc-Trap®_A beads and pipette 25 μL bead slurry into 500 μL ice-cold dilution buffer. Centrifuge at 2.500x g for 2 min at +4 °C. Discard supernatant and repeat wash twice.
- Harvest cells
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10-cm dish) expressing a 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. After washing:
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 supernatant to a pre-cooled tube. Add 300 μL dilution buffer to supernatant. 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.
5. Add diluted supernatant (step 3) to equilibrated Myc-Trap®_A beads (step 4). If required, save 50 μL of diluted lysate for immunoblot analysis. Tumble end-overend for 1 hour at 4 °C.
6. Centrifuge at 2.500x g for 2 min at +4 °C. If required, save 50 μL supernatant for immunoblot analysis. Discard remaining supernatant.
7. Resuspend Myc-Trap®_A beads in 500 μL ice-cold dilution buffer. Centrifuge at 2.500x g for 2 min at +4 °C. Discard supernatant and repeat wash twice.
optional: Increase salt concentration in the second washing step up to 500 mM.
8. Resuspend Myc-Trap®_A beads in 100 μL 2x SDS-sample buffer.
9. Boil resuspended Myc-Trap®_A beads for 10 min at 95 °C to dissociate immunocomplexes from Myc-Trap®_A beads. Myc-Trap®_A beads can be collected by centrifugation at 2.500x g for 2 min at 4 °C and SDS-PAGE is performed with the supernatant.
10. Optional instead of steps 8 and 9: Reconstitute Myc peptide (code yp-1) in water to a final concentration of 2 mg/ ml. Add ~25 μg Myc peptide in 50-100 μL in dilution buffer to Myc-Trap®_A beads. Mix for 15 min at RT. Centrifuge at 2500xg for 2 min and carefully transfer supernatant to a new tube. To increase elution efficiency this step can be repeated.
Note: Use our spin column protocol for easy elution
- Nur für Forschungszwecke einsetzbar
- 20 % EtOH
- Do not freeze.
- 4 °C
- 12 months
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