GST-Trap

Details for Product No. ABIN1082203, Supplier: Log in to see
Antigen
  • GST
  • GST 13-13
  • GST13
  • GST13-13
  • GSTK1-1
  • hGSTK1
  • GSTS
  • GSTS1-1
  • PGD2
  • PGDS
  • H-PGDS
  • Ptgds2
  • 1500002K10Rik
  • Gst
  • glutathione S-transferase kappa 1
  • hematopoietic prostaglandin D synthase
  • microsomal glutathione S-transferase 1
  • GSTK1
  • HPGDS
  • Hpgds
  • Mgst1
Reactivity
Tag
4
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
Host
Camelidae
Antibody Type
Recombinant Antibody
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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Purpose GST-Trap is a highly specific and robust GST-binding protein for biochemical analysis of GST fusion proteins and their interacting partners.
Sample Type Cell Extracts
Characteristics 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.
Purification purified protein
Components GST-Trap uncoupled
Material not included Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
Alternative Name GST (GST ELISA Kit Abstract)
Background 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.
Research Area Tags/Labels, Metabolism, Immunology
Application Notes 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.
Assay Time 1.5 h
Protocol
  • 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
Reagent Preparation

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

Assay Procedure
  • 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.
Restrictions For Research Use only
Format Liquid
Concentration 1 mg/ml
Buffer 1 x PBS,0.01% Sodium azide
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
Immunoprecipitation (IP) image for GST-Trap (ABIN1082203) Pulldown of GST using the GST-Trap Immunoprecipitations (IP) of GST from protein extr...
Western Blotting (WB) image for GST-Trap (ABIN1082203) Western Blot Analysis of purified GST: 10, 25, 50, 100, 250, 50 ng GST, detected with...
Western Blotting (WB) image for GST-Trap (ABIN1082203) Protein lysate of E.coli cells expressing GST was spearated by SDS-Page and analyzed ...
Western Blotting (WB) image for GST-Trap (ABIN1082203) Pulldown of GST using the GST-Trap Immunoprecipitations (IP) of GST from protein extr...
Western Blotting (WB) image for GST-Trap (ABIN1082203) Western Blot Analysis of purified GST: 10, 25, 50, 100, 250, 50 ng GST, detected with...
Western Blotting (WB) image for GST-Trap (ABIN1082203) Protein lysate pf E.coli cells expressing GST was spearated by SDS-Page and analyzed ...