Mdm4 / HdmX-Trap A

Details for Product No. ABIN1889481, Supplier: Log in to see
Antigen
  • mdmx
  • HDMX
  • MDMX
  • MRP1
  • wu:fa09h09
  • wu:fi33d10
  • 4933417N07Rik
  • AA414968
  • AL023055
  • AU018793
  • AU021806
  • C85810
  • Mdmx
  • Mdm4 p53 binding protein homolog
  • Mdm4 p53 binding protein homolog (mouse)
  • transformed 3T3 cell double minute 4 homolog (mouse)
  • transformed mouse 3T3 cell double minute 4
  • mdm4
  • MDM4
  • Mdm4
Alternatives
Human MDM4-binding Protein ELISA Kit
Epitope
AA 1-129
Reactivity
Human
5
3
3
Host
Camelidae
Conjugate
Agarose Beads
Application
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)
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Supplier Product No.
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Purpose The Mdm4/ HdmX-Trap is a high quality Mdm4/ HdmX-binding protein coupled to a monovalent matrix (agarose particles) for biochemical analysis of Mdm4/ HdmX and interacting partners.
Sample Type Cell Extracts
Specificity Species-Reactivity: tested on human, mouse, hamster
Characteristics Mdm4/ HdmX-Trap is excellent for fast and efficient one-step isolation of Mdm4/ HdmX and its interacting factors from cellular extract. Isolated Mdm4/ HdmX protein may be used further for immunoblot analysis or mass spectrometry.
Mdm4/ HdmX-Trap utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
Components Mdm4/ HdmX-Trap coupled to agarose beads
Material not included Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
Alternative Name Mdm4 (MDM4 ELISA Kit Abstract)
Background Double minute 4 protein (Mdm4/ HdmX/ MdmX) plays a role in cell cylce control and apoptosis by inhibition of p53 and p73.
Comment

Bead size ~ 90 µm
Note that binding of Mdm4/ HdmX-Trap interferes with binding of p53 to Mdm4/ HdmX.

Assay Time 1.5 h
Protocol
  • Robust and versatile tool for biochemical analyses of Mdm4/ HdmX 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)
Sample Collection 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.
Assay Procedure

Equilibrate beads

  • 4. Vortex Mdm4/ HdmX_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 Mdm4/ HdmX_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 Mdm4/ HdmX_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 Mdm4/ HdmX_A beads in 100 µL 2x SDS-sample buffer.
  • 9. Boil resuspended Mdm4/ HdmX_A beads for 10 min at 95°C to dissociate immunocomplexes from Mdm4/ HdmX_A beads. Mdm4/ HdmX_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.

Restrictions For Research Use only
Buffer Storage buffer: 20 % EtOH
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
Immunoprecipitation (IP) image for Mdm4 / HdmX-Trap A (ABIN1889481) Pulldown of Mdm4/HdmX using the Mdm4/HdmX-Trap:Immunoprecipitation (IP) of Mdm4/HdmX ...
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