GST-Trap M

Details zu Produkt Nr. ABIN1082190, Anbieter: Anmelden zum Anzeigen
Antigen
Reaktivität
Schistosoma japonicum
3
3
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
Wirt
Camelid (Camelidae)
Antikörpertyp
Recombinant Antibody
Konjugat
Magnetic Particles
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
Anmelden zum Anzeigen
Hersteller Produkt- Nr.
Anmelden zum Anzeigen
Verwendungszweck GST-Trap is a highly specific and robust GST-binding protein coupled to a monovalent matrix (magnetic particles) for biochemical analysis of GST fusion proteins and their interacting partners.
Proben Cell Extracts
Spezifität Binding capacity: 10 µL GST-Trap_M slurry binds 0.25 - 0.5 µg of GST
Produktmerkmale 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.
Bestandteile GST-Trap coupled to magnetic particles
Benötigtes Material Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
Andere Bezeichnung GST (GST ELISA Kit Abstract)
Hintergrund Fusion of proteins to Glutathione S-Transferase (GST) of Schistosoma japonicum is a common technique to increase solubility and expression level of a protein. Furthermore, GST- tagged fusion proteins are often used in protein-protein interaction studies. Both approaches require highly specific tools to isolate and detect GST fusion proteins
Forschungsgebiet Tags/Labels, Metabolism, Immunology
Applikationshinweise For biochemical analyses including mass spectroscopy and enzyme activity measurements these GST-fusion proteins and their interacting factors can be isolated fast and efficiently (one step) via immunoprecipitation using the GST-Trap. Since the interaction is mediated by a small GST-binding protein coupled to magnetic particles the GST-Trap_M enables purification of any protein of interest fused to GST.
Kommentare

Bead size 0.5 - 1 µm

Testdauer 1.5 h
Protokoll
  • Robust and versatile tool for biochemical analyses of GST-fusion proteins
  • Short incubation times (5 - 30 min)
  • Low unspecific binding
  • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
  • No contaminating heavy and light chains of conventional antibodies
Aufbereitung der Reagenzien

Suggested buffer composition

  • Lysis buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
  • Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
  • Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
  • Elution buffer: 200 mM glycine pH 2.5

Testdurchführung
  • 1. For one immunoprecipitation reaction resuspend cell pellet of 1 mL bacteria culture expressing GST in 100 - 200 µL lysis buffer by pipetting.
    optional: add 1 mM PMSF and 0.1 mg/ml lysozyme to lysis buffer
  • 2. Rotate tube for 1h at 4°C.
  • 3. Sonify cell pellet to disrupt bacterial membrane.
  • 4. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C.
  • 5. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500 µL. Discard pellet.For immunoblot analysis dilute 30 µL cell lysate with 10 µL 4x SDS-sample buffer(-> refer to as input).
  • 6. Equilibrate GST-Trap_M beads in dilution buffer. Resuspend magnetic particles by vortexing and transfer calculated volume (20 - 30 µL) in a new reaction cup with 250 µL ice cold dilution buffer. Magnetically separate until supernatant is clear and wash twice with 250 µL of cold dilution buffer.
  • 7. Add cell lysate to equilibrated GST-Trap_M beads and incubate the GST-Trap_M beads with the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C.
    note: during incubation of protein sample with the GST-Trap_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix
  • 8. Magnetically separate until supernatant is clear. For western blot analysis dilute 30 µL supernatant with 10 µL 2x SDS-sample buffer (-> refer to as non-bound). Discard remaining supernatant
  • 9. Wash beads two times with 500 µL ice cold wash buffer.
    optional: increase salt concentration in the second washing step up to 500 mM
  • 10. Resuspend GST-Trap_M beads in 100 µL 2x SDS-Sample buffer or go to step 11.
  • 11. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be collected by centrifugation at 2.500x g for 2 minutes at 4°C and SDS-PAGE is performed with the supernatant (-> refer to as bound).
  • 12. optional: 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 fresh cup 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
Konzentration 2.5 mL resin
Buffer 1 x PBS,0.01% Sodium azide
Konservierungsmittel Sodium azide
Vorsichtsmaßnahmen This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handhabung Do not freeze.
Lagerung 4 °C
Haltbarkeit 12 months
Bilder des Herstellers
Western Blotting (WB) image for GST-Trap M (ABIN1082190) Pulldown of GST using the GST-Trap Immunoprecipitations (IP) of GST from protein extr...
Western Blotting (WB) image for GST-Trap M (ABIN1082190) Western Blot Analysis of purified GST: 10, 25, 50, 100, 250, 50 ng GST, detected with...
Western Blotting (WB) image for GST-Trap M (ABIN1082190) Protein lysate pf E.coli cells expressing GST was spearated by SDS-Page and analyzed ...
Haben Sie etwas anderes gesucht?