MK2-Trap A Kit

Details zu Produkt Nr. ABIN1889471, Anbieter: Anmelden zum Anzeigen
Antigen
  • mapkapk2
  • wu:fb30e10
  • wu:fc56c10
  • wu:fi03a01
  • MGC78852
  • MAPKAPK2
  • AA960234
  • MAPKAP-K2
  • MK-2
  • MK2
  • Rps6kc1
  • mitogen-activated protein kinase-activated protein kinase 2a
  • mitogen-activated protein kinase-activated protein kinase 2 L homeolog
  • mitogen-activated protein kinase-activated protein kinase 2
  • MAP kinase-activated protein kinase 2
  • mapkapk2a
  • mapkapk2.L
  • MAPKAPK2
  • Mapkapk2
Alternativen
Human MAPKAP Kinase 2 ELISA Kit
Reaktivität
Human, Maus, Hamster
11
11
9
2
Wirt
Camelid (Camelidae)
Antikörpertyp
Recombinant Antibody
Konjugat
Agarose Beads
Applikation
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
Optionen
Hersteller
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Hersteller Produkt- Nr.
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Verwendungszweck The MK2-Trap is a high quality MK2-binding protein coupled to a monovalent matrix (agarose particles) for biochemical analysis of MK2 and interacting partners.
Proben Cell Extracts
Spezifität Posttranslational Modifications: MK2-Trap recognizes unphosphorylated MK2 and Phospho-MK2 (Thr222). Specificity on Phospho-MK2 (Thr334) was not tested.
Produktmerkmale MK2-Trap is excellent for fast and efficient one-step isolation of MK2 and its interacting factors from cellular extract. Isolated MK2 protein may be used further for immunoblot analysis, mass spectrometry, and kinase assays.
MK2-Trap utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
Bestandteile MK2-Trap coupled to agarose beads
Lysis buffer (CoIP) 40 mL
10x RIPA buffer 2 mL
Dilution buffer 150 mL
Wash buffer 40 mL
Elution buffer 3 mL
Andere Bezeichnung MK2 (MAPKAPK2 ELISA Kit Abstract)
Hintergrund Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 / MK2) belongs to the family of serine/threonine kinases. In response to cellular stress it is phosphorylated and activated by MAP kinase p38.
Kommentare

Bead size ~ 90 µm

Testdauer 1.5 h
Protokoll
  • Robust and versatile tool for biochemical analyses of MK2 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)
Probennahme Harvest cells:
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) is recommended. To harvest adherent cells, aspirate growth medium, add 1 ml ice-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.
Testdurchführung

Equilibrate beads

  • 4. Vortex MK2_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.

Bind proteins:
  • 5. Add diluted lysate (step 3) to equilibrated MK2_A 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. Centrifuge at 2.500x g for 2 min at +4°C. If required, save 50 µL supernatant for immunoblot analysis. Discard remaining supernatant.

Wash beads:
  • 7. Resuspend MK2_A beads in 500 µL 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.

Elute proteins:
  • 8. Resuspend MK2_A beads in 100 µL 2x SDS-sample buffer.
  • 9. Boil resuspended MK2_A beads for 10 min at 95°C to dissociate immunocomplexes from MK2_A beads. MK2_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: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. 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.

Beschränkungen Nur für Forschungszwecke einsetzbar
Buffer Storage buffer: 20 % EtOH
Handhabung Do not freeze.
Lagerung 4 °C
Haltbarkeit 12 months
Bilder des Herstellers
Immunoprecipitation (IP) image for MK2-Trap A Kit (ABIN1889471) Pulldown of MK2 using the MK2-Trap: Immunoprecipitations (IP) of MK2 from a protein e...
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