p53-Trap A

Details zu Produkt Nr. ABIN1889474, Anbieter: Anmelden zum Anzeigen
Antigen
  • BCC7
  • LFS1
  • P53
  • TRP53
  • p53
  • Trp53
  • brp53
  • drp53
  • etID22686.5
  • fb40d06
  • wu:fb40d06
  • zgc:111919
  • Tp53
  • bbl
  • bfy
  • bhy
  • p44
  • tp53
  • Xp53
  • TP53
  • tumor protein p53
  • cellular tumor antigen p53
  • transformation related protein 53
  • tumor protein p53 L homeolog
  • transformation related protein 53, pseudogene
  • Wistar clone pR53P1 p53 pseudogene
  • TP53
  • CpipJ_CPIJ002758
  • Tp53
  • tp53
  • Trp53
  • tp53.L
  • Trp53-ps
  • p53-ps
Epitop
AA 1-81
Reaktivität
Human
78
33
30
4
4
4
4
3
3
3
3
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 p53-Trap is a high quality p53-binding protein coupled to a monovalent matrix (agarose particles) for biochemical analysis of p53 and interacting partners.
Proben Cell Extracts
Spezifität Species-Reactivity: tested on human
Produktmerkmale p53-Trap is excellent for fast and efficient one-step isolation of p53 and its interacting factors from cellular extract. Isolated p53 protein may be used further for immunoblot analysis or mass spectrometry.
p53-Trap utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
Bestandteile p53-Trap coupled to agarose beads
Benötigtes Material Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
Andere Bezeichnung p53 (TP53 ELISA Kit Abstract)
Hintergrund The tumor suppressor protein p53 plays a major role in cell cycle control, apoptosis and senescence.
Applikationshinweise p53-Trap is excellent for fast and efficient one-step isolation of p53 and its interacting factors from cellular extract. Isolated p53 protein may be used further for immunoblot analysis or mass spectrometry.
p53-Trap utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
Kommentare

Bead size ~ 90 µm

Testdauer 1.5 h
Protokoll
  • Robust and versatile tool for biochemical analyses of p53 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 p53_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 p53_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 p53_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 p53_A beads in 100 µL 2x SDS-sample buffer.
  • 9. Boil resuspended p53_A beads for 10 min at 95°C to dissociate immunocomplexes from p53_A beads. p53_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 p53-Trap A (ABIN1889474) Pulldown of p53 using the p53-Trap: Immunoprecipitations (IP) of p53 from a protein e...
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