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 제품안내  면역학   Cell Signaling  G-Protein Signaling

G-LISA Kit

G-Protein G-LISA Kit은 세포나 조직내의 G단백질 (Ras, RhoA, Rac, Cdc42)의 activation level을 측정해주는 제품으로, GST bead를 사용하는 기존 방식과 달리, G-Protein effector가 96well plate에 코팅되어 있어 샘플내 active 단백질이 반응되어 붙으면, 이를 항체 및 HRP로 측정하는 방식입니다.

Cytoskeleton
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특징
  • 각 G-Protein effector로 conjugation된 96well plate를 이용하여, 샘플내 active G-Protein이 반응하여 붙으면, 이것을 1,2차 항체를 이용하여 detection 합니다.
  • HRP를 이용하여, colorimetric 혹은 Luminescent 방법으로 detection 합니다.
  • 이틀이 걸리는 기존 Pull down 방식에 비해, 단 3시간이내에 결과를 얻을수 있습니다.
  • 적은양의 샘플로도 측정이 가능합니다. (5-25ug cell protein)
  • Yield가 높고, 결과가 정확합니다.
  • High thoughput screens시 이용하면 편리합니다.
원리
G-Protein G-LISA Kit Protocol
기존 Pull down 방식과 G-LISA의 차이
 Pull down assayG-LISA™
Assay Time 10-12 h
(2 days)
< 3h
Cell material per assay 1-2 mg protein
(100 mm plate)
1-50 μg protein
(12 to 96-well plate)
Lysate clarification needed Yes No
Sample handling Up to 10 samples Up to 96 samples(or more)
Quantitative data Semi Yes
High throughput compatible No Yes
G-LISA를 이용한 chemiluminesce방식과 absorbance방식의 차이
 Luminometric detectionColorimetric detection
Assay Time  
Cell material per assay 1-25 ug protein 2-50 ug protein
Measurement parameters Luminometer - high gain and 100 ms read per well Spectrophotometer 490 nm
Detection limit* 0.25 ng small G-protein 0.50 ng small G-protein
Linear range of detection* 0.025-1.0 ng 0.05-2.0 ng
cv of 8 replicates 16% 12%
* Determined by titrating the amount of recombinant Rho added to the wells
적용
Figure 1. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA kit BK121. Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA. 25 ug of lysates were subjected to the G-LISA assay. Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates).Blue = Serum Starved, Yellow = Activated.
Figure 2. Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04) using the RhoA G-LISA Activation Assay (BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 ug/ml of CT04 for 4 h in serum free medium at 37ºC, then activated with 100 ug/ml calpeptin for 10 min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 ug/ml CT04 for 4 h results in almost complete (90%) inhibition of RhoA activity.
Figure 3. Time course of RhoA activation by 10 ug/ml LPA, 100 ug/ml calpeptin (Cat.# CN01) and 1 ug/ml CNF (Cat.# CN03) in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with activator for the times shown in the graph and RhoA activation was analyzed with G-LISA kit BK121. LPA stimulation leads to a rapid and transient activation of RhoA, which peaks at 2-3 min and quickly declines to basal levels. Activation by calpeptin leads to a signal increase between 10-30 min which declines to basal after 60 min. Activation by CNF leads to signal increase after 60 min which is stable for up to 12 h.
종류
  • Ras G-LISA Activation Assay Kit (Cat# BK131)
    • 세포나 조직내 Ras Activation을 측정할 수 있는 제품입니다.
    • Ras-GTP affinity wells을 이용하여 이에 반응된 active Ras를 detection 합니다.
    • Colorimetric 방법으로 측정됩니다.
    • 제품의 구성:
      • 96 Ras-GTP affinity wells (divisible into 12 strips of 8 wells each)
      • Lysis buffer
      • Binding buffer
      • Antigen presenting buffer
      • Wash buffer
      • Antibody dilution buffer
      • Anti-Ras antibody
      • HRP-labeled secondary antibody
      • Positive control Ras protein
      • Protease inhibitor cocktail (Cat. # PIC02)
      • Absorbance detection reagents
      • Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
    • 적용
      • Ras signaling pathway studies
      • Ras activation assays with primary cells
      • Studies of Ras activators and inactivators
      • Ras activation assays with limited material
      • High throughput screens for Ras activation
    • 사용예
      Ras activation by EGF measured by G-LISA™. HeLa cells were serum starved(ss) for 24 h and treated with EGF(100 ng/ml for 2 min). 25, 12.5, 5, 1.25 ug of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm. Data are background subtracted
  • RhoA G-LISA™ Activation Assay Kit (Cat# BK124, BK121)
    • 세포나 조직내 Activated RhoA를 측정할 수 있는 제품입니다.
    • Rho-GTP affinity wells을 이용하여 이에 반응된 active RhoA를 detection 합니다.
    • Colorimetric (BK124) 혹은 Luminescent (BK121) 방법으로 측정이 가능합니다.
    • 제품의 구성:
      • 96 Rho-GTP affinity wells (divisible into 12 strips of 8 wells each for multiple uses)
      • Lysis buffer
      • Binding buffer
      • Antigen presenting buffer
      • Wash buffer
      • Antibody dilution buffer
      • Anti-RhoA antibody
      • HRP-labeled secondary antibody
      • Positive control RhoA protein
      • Protease inhibitor cocktail
      • Absorbance detection reagents (or Luminescence detection reagents)
      • Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
    • 적용
      • Rho signaling pathway studies
      • Rho activation assays with primary cells
      • Studies of Rho activators and inactivators
      • Rho activation assays with limited material
      • High throughput screens for Rho activation
    • Product Citations
      • Warren JC, Rutkowski A. and L. Cassimeris. 2006. Infection with replication-deficient adenovirus induces changes in the dynamic instability of host cell microtubules. Mol. Biol. Cell, 17, p3557-3568.
      • Woods A and Beier F. 2006. RhoA/ROCK signaling regulates chondrogenesis in a context-dependent manner. J Biol Chem. May 12; 281(19):13134-40.
      • Higashibata A, Imamizo-Sato M, Seki M, Yamazaki T. and Ishioka N. 2006. Influence of simulated microgravity on the activation of small GTPase Rho involved in cytoskeletal formation - molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor. BMC Biochemistry, 7, (19), p1-9.
    • 사용예
      Figure 1. RhoA activation by calpeptin measured by G-LISA™ kit BK124. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with calpeptin (Cal; 0.1 mg/ml for 30 min) or DMSO carrier only (SS). 10μg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.
      Figure 2. Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04) using the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 μg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100μg/ml calpeptin for 10min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 μg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.
      Figure 3. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA™ kit BK121. Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA. 25 μg of lysates were subjected to the G-LISA™ assay. Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates). Numbers above LPA bars correspond to fold activation compared to the control serum starved samples.
  • Rac1 or Rac1,2,3 G-LISA Activation Assay Kit (Cat# BK126, BK125)
    • 세포나 조직내 Activated Rac1을 측정할 수 있는 제품입니다.
    • Rac-GTP affinity wells을 이용하여 이에 반응된 active Rac을 detection 합니다.
    • 제품의 구성:
      • 96 Rac-GTP affinity wells (divisible into 96 individual wells)
      • Lysis buffer
      • Binding buffer
      • Antigen presenting buffer
      • Wash buffer
      • Antibody dilution buffer
      • Anti-Rac1 antibody
      • HRP-labeled secondary antibody
      • Positive control Rac1 protein
      • Protease inhibitor cocktail
      • Absorbance detection reagents (or Luminescence detection reagents)
      • Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
    • 적용
      • Rac1 signaling pathway studies
      • Rac1 activation assays with primary cells
      • Studies of Rac1 activators and inactivators
      • Rac1 activation assays with limited material
      • High throughput screens for Rac1 activation/inhibition
    • Product Citations
      • Ramirez SH, Heilman D, Morsey B, Potula R, Haorah J and Persidsky Y. Activation of Peroxisome Proliferator-Activated Receptor (PPAR) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes. J. Immunology, 2008, 180, p1854-1865.
      • Pontow S, Harmon B, Campbell N, and Ratner L. Antiviral Activity of a Rac GEF inhibitor Characterized with a Sensitive HIV/SIV Fusion Assay. Virology. 2007 November 10; 368(1): 1-6.
      • Zhou X, Tian F, Sandze J, Cao R, Flaberg E, Szekely L, Cao Y, Ohlsson C, Bergo M, Bore' n J and Akyu¨ rek LM. Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development. PNAS, March 6th, 2007, 104, (10), p3919-3924.
      • Schlegel N, Burger S, Golenhofen N, Walter U, Drenckhahn D, and Waschke J. The role of VASP in the regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization. Am J Physiol Cell Physiol (November 7, 2007). doi:10.1152/ajpcell.00273.2007
    • 실험예
      Figure 1. Rac1 activation by EGF measured by G-LISA™ kit BK126. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with EGF (10ng/ml for 2.5 min) or buffer only (SS). 10 μg of cell lysates were subjected to the G-LISA™ assay. Luminescence measured over 100 milli-second.
      Figure 2. Rac activation by EGF measured by G-LISA™. Swiss 3T3 cells were serum starved (SS) for 24 h and treated with EGF (10 ng/ml for 2 min). 60, 30, 15, 7.5, 2.5 μg of cell lysates were subjected to the G-LISA™ assay. Luminescence was measured over 10 milli-seconds. 500 μg of the same lysates were subjected to the traditional PAK pull-down assay (shown in inset, Cat.# BK035).
  • Cdc42 G-LISA Activation Assay Kit (Cat# BK127)
    • 세포나 조직내 Activated Cdc42를 측정할 수 있는 제품입니다.
    • Cdc42-GTP affinity wells을 이용하여 이에 반응된 active Cdc42를 detection 합니다.
    • 제품의 구성:
      • 96 Cdc42-GTP affinity wells (divisible into 12 strips of 8 wells each)
      • Lysis buffer
      • Antigen presenting buffer
      • Wash buffer
      • Antibody dilution buffer
      • Anti-Cdc42 antibody (Cat.# ACD03)
      • HRP-labeled secondary antibody
      • Positive control Cdc42 protein
      • Protease inhibitor cocktail
      • Color development reagents
      • Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
    • 적용
      • Cdc42 signaling pathway studies
      • Cdc42 activation assays with primary cells
      • Studies of Cdc42 activators and inactivators
      • Cdc42 activation assays with limited material
      • High throughput screens for Cdc42 activation
    • Product Citations
      • Warren JC, Rutkowski A. and L. Cassimeris. 2006. Infection with replication-deficient adenovirus induces changes in the dynamic instability of host cell microtubules. Mol. Biol. Cell, 17, p3557-3568.
      • Woods A and Beier F. 2006. RhoA/ROCK signaling regulates chondrogenesis in a context-dependent manner. J Biol Chem. May 12; 281(19):13134-40.
    • 실험예
      Figure 1. Cdc42 activation by EGF measured by the Cdc42 G-LISA™ Activation Assay. Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (100 ng/ml for 2 min). Cell lysates (8, 17, 35 μg) were subjected to the G-LISA™ assay. Data was read at 490 nm. Numbers on top the yellow columns indicate the fold increase in signal caused by EGF activation, you will notice the ratio remains the same with different protein loadings indicating good linearity with different protein loadings. 500 μg of the same lysates were subjected to the traditional PAK pull-down assay (Cat.# BK034) with similar results.
      Figure 2. Time course of Cdc42 activation using EGF at 10, 50 and 100ng/ml. Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (10, 50 and 100 ng/ml for1.5, 3.0, 6.0, 10 and 30 min). Cell lysates subjected to the G-LISA™ assay and OD was read at 490 nm. The “controlled state” serum starved value (0.22) was subtracted from these samples prior to plotting. At 100 ng/ml the total activation was 2.1 fold or 110% over the controlled state at 1.5 min. This type of analysis can be performed in one afternoon.
  • Rac1 G-LISA™ Activation Assay (Colorimetric Based) (Cat# BK128)
    • 기존 pull down 방식에 비해 훨씬 빠르고 쉽게 Rac1 activation을 측정할 수 있습니다.
    • Rac1에 specific 합니다.
    • 3시간안에 결과를 얻을수 있습니다.
    • Colorimetric 방법으로 측정됩니다.
    • 소량의 샘플로 실험이 가능합니다. (10-50 ug protein (3 cm plate))
    • 제품의 구성:
      • 96 individual Rac1-GTP binding wells
      • Anti-Rac1 antibody (Cat. # ARC03)
      • Secondary Antibody - HRP
      • Rac1 control protein
      • Cell Lysis Buffer
      • Wash buffer
      • Antigen Presenting Buffer
      • Antibody Dilution Buffer
      • HRP Detection and Stop Reagents
      • Precision Red™ Advanced Protein Assay (Cat. # ADV02)
      • Protease Inhibitor Cocktail (Cat. # PIC02)
  • RalA G-LISA™ Activation Assay (Colorimetric Based) (Cat# BK129)
    • 기존 pull down 방식에 비해 훨씬 빠르고 쉽게 RalA activation을 측정할 수 있습니다.
    • RalA에 specific 합니다.
    • 3시간안에 결과를 얻을수 있습니다.
    • Colorimetric 방법으로 측정됩니다.
    • 소량의 샘플로 실험이 가능합니다. (10-50 ug protein (3 cm plate))
    • 제품의 구성:
      • 96 individual Ral-GTP binding wells
      • Anti-RalA monoclonal antibody
      • Secondary Antibody - HRP
      • RalA control protein
      • Ral Binding Buffer
      • Cell Lysis Buffer
      • Wash buffer
      • Antigen Presenting Buffer
      • Antibody Dilution Buffer
      • HRP Detection and Stop Reagents
      • Precision Red™Advanced Protein Assay (Cat. # ADV02)
      • Protease Inhibitor Cocktail (Cat. # PIC02)
  • Total RhoA ELISA Kit (Cat# BK150)
      • 세포나 조직 내 Total RhoA와 Active RhoA의 양을 측정하는 제품입니다.
      • 제품의 구성:
        • 96 well Total Rho binding plate (12 strips of 8 wells each)
        • Anti-RhoA antibody
        • Secondary Antibody - HRP
        • Rho control protein
        • Dilution Buffer
        • Wash Buffer (PBST)
        • Antigen Presenting Buffer
        • HRP Detection Reagents
      • 적용:
        • Explore RhoA pathway mechanisms in diverse research fields
        • Normalize RhoA activation levels in transfection assays
        • Analyse link between RhoA activation and cancers
        • Quickly quantitate normalized RhoA activation data with less than 25 ug lysate
      • RhoA ELISA와 Western blot 비교
    CharacteristicComparison
    Western blottingRhoA ELISA
    Assay Time 6-8 h 5 h
    Cell material per assay 10-50 μg protein 3-30 μg protein
    Compatibility with different lysis buffers Incompatible with high salt buffers Compatible with most buffers tested
    Sample handling 2-20 samples 2-96 samples (or more)
    Quantitative Data Semi Yes (Numerical readouts and fewer sample handling steps make this assay more quantitative)
    High throughput compatible No Yes