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 제품안내  면역학   Cell Signaling  G-Protein Signaling

G-Protein Biochem Kit

G-Protein Biochem Kit은 세포나 조직내의 G단백질 (Ras, RhoA, Rac, Cdc42)의 activation level을 측정해주는 제품으로, G-Protein effector로 conjugation된 glutathione-sepharose (GST) bead가 타겟 G-Protein의 active GTP에 반응함으로써 active한 G단백질만 침전시켜 얻어내는 전통적인 pulldown assay 방식입니다.

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특징
  • 각 G-Protein effector로 conjugation된 glutathione-sepharose (GST) bead를 이용하여 세포나 조직내 타겟 G-Protein을 침전시킵니다.
  • 침전된 active G-Protein은 전기영동후, kit에서 제공되는 항체를 이용하여 Western Blotting으로 측정합니다.
종류 및 특징
  • RhoA Activation Assay Biochem Kit (Cat# BK036)
    • 세포나 조직내 Activated RhoA를 측정할 수 있는 제품입니다.
    • GST-tagged Rhotekin-RBD protein을 이용하여 침전시킵니다.
    • 제품의 구성:
      • GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # RT02)
      • RhoA-specific monoclonal antibody (Cat. # ARH01)
      • His-tagged RhoA protein (Cat. # RH01)
      • GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
      • GDP
      • Cell lysis Buffer
      • Wash Buffer
      • Loading Buffer
      • STOP Buffer
      • Protease inhibitor cocktail
    • 적용
      • Analysis of in vivo RhoA activation
    • 실험예
      • The RhoA activation assay was tested by loading the RhoA protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded RhoA is very efficiently precipitated while very little GDP-loaded RhoA is precipitated (Fig. 1)
        Figure 1. Results from BK036 RhoA activation assay. Activated Rac was precipitated and detected in a Western blot using kit BK036. The first lane shows a 50 ng recombinant His-tagged RhoA standard (Rec. His-RhoA). The following lanes shows the pulldown of inactive, GDP-loaded, (RhoA-GDP PD) or active, GTPγS-loaded, Rac (RhoA-GTP PD) from equal amounts of cell lysates.
         
  • Ras Activation Assay Biochem Kit (Cat# BK008)
    • 세포나 조직내 Activated Ras를 측정할 수 있는 제품입니다.
    • GST tagged Raf1-RBD protein을 이용하여 침전시킵니다.
    • 제품의 구성:
      • GST tagged Raf1-RBD protein bound to colored glutathione agarose beads (Cat. # RF02)
      • p21 Ras monoclonal antibody
      • Cell lysis Buffer
      • Protease inhibitor cocktail
      • Wash Buffer
      • Loading Buffer
      • STOP Buffer
      • GTPγS (non-hydrolyzable GTP analog, Cat. # BS01)
      • GDP
    • 적용
      • Analysis of in vivo RhoA activation
  • Rac1 Activation Assay Biochem Kit (Cat# BK035)
    • 세포나 조직내 Activated Rac1을 측정할 수 있는 제품입니다.
    • GST-tagged PAK-PBD protein을 이용하여 침전시킵니다.
    • 제품의 구성:
      • GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
      • Rac1 monoclonal antibody (Cat. # ARC03)
      • His-tagged Rac1 protein (Cat. # RC01)
      • GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
      • GDP
      • Cell lysis Buffer
      • Wash Buffer
      • Loading Buffer
      • STOP Buffer
      • Protease inhibitor cocktail
    • 적용
      • Analysis of in vivo Rac1 activation levels.
      • Detection of compounds and proteins that enhance Rac1 activity.
      • Detection of compounds and proteins that inhibit Rac1 activity.
    • 실험예
      • The Rac1 activation assay was tested by loading the Rac1 protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded Rac1 is very efficiently precipitated while very little GDP-loaded Rac1 is precipitated (Fig. 1)
        Figure 1. Results from BK034 Cdc42 activation assay. Activated Cdc42 was precipitated and detected in a Western blot using kit BK034. The first lane shows a 50 ng recombinant His-tagged Cdc42 standard (Rec. His-Cdc42). The following lanes shows the pulldown of iinactive, GDP-loaded, (Cdc42-GDP PD) or active, GTPγS-loaded, Cdc42 (Cdc42-GTP PD) from equal amounts of cell lysates.
         
  • RalA Activation Assay Biochem Kit (BK040)
    • 세포나 조직내 Activated RalA를 측정할 수 있는 제품입니다.
    • 제품의 구성:
      • Active Ral Affinity beads
      • Anti-RalA monoclonal antibody
      • His-Ral A control Protein
      • Cell Lysis Buffer
      • Wash Buffer
      • Loading Buffer
      • STOP Buffer
      • GTPγS stock: (non-hydrolysable GTP analog)
      • GDP stock
      • Protease inhibitor cocktail
      • Anhydrous DMSO
    • 실험예
      Figure 1. RalBP1-RBD bead pulldown Assays. Rat2 cells were grown to 70% confluency in DMEM media supplemented with 10% fetal calf serum (FCS) prior to 48h growth in serum free media. Cell lysates were processed as described in Section VI of this manual. Active RalA was purified from 400 μg of cell lysates by incubating lysates with 10 μg of RalBP1-RBD beads as described in section VI. All bead samples were resuspended in 10 μl of 2x sample buffer and run on a 4-20% SDS gel. Protein was transferred to PVDF, probed with a 1:500 dilution of anti-RalA and processed for chemiluminescent detection of RalA as described in Section VI: STEP 4. Lane 1: 20 ng recombinant RalA-His control protein; Lane 2: bead bound RalA from lysates of cells treated for 2 minutes with Epidermal growth factor (100mg/ml); Lane 3: bead bound RalA from lysates of cells grown in serum free media for 48h. Densitometric analysis of chemiluminescent signal showed that RalA activation in EGF tretaed cells was 2 fold above RalA activation in serum starved cells.
       
  • Cdc42 Activation Assay Biochem Kit (BK034)
    • 세포 내 Cdc42 활성을 측정하는 제품입니다.
    • 제품의 구성:
      • GST-tagged PAK-PBD protein on colored agarose beads
      • Cdc42 polyclonal antibody
      • His-tagged Cdc42 protein
      • GTPγS: (non-hydrolyzable GTP analog)
      • GDP
      • Cell lysis Buffer
      • Wash Buffer
      • Loading Buffer
      • STOP Buffer
      • Protease inhibitor cocktail
    • 적용
      • Analysis of in vivo Cdc42 activation
    • 실험예
      Figure 1. Results from BK034 Cdc42 activation assay.
      Activated Cdc42 was precipitated and detected in a Western blot using kit BK034. The first lane shows a 50 ng recombinant His-tagged Cdc42 standard (Rec. His-Cdc42). The following lanes shows the pull-down of inactive, GDP-loaded Cdc42 (Cdc42-GDP PD) or active, GTPγS-loaded Cdc42 (Cdc42-GTP PD) from equal amounts of cell lysates.