NewEast Biosciences丨Gαs 活性检测试剂盒丨武汉试剂盒厂家

武汉费斯德生物科技有限公司是美国neweast biosciences在中国的办事处。

neweast biosciences在十二年前率先研发俩种独特的抗体。这俩种抗体仅仅识别活性的gtp酶或者突变的oncogene。

 gtp酶涉及(1)响应细胞表面受体激活的信号转导，包括跨膜受体，例如介导味觉、嗅觉和视觉的那些，（2）核糖体的蛋白质生物合成，（3）调节细胞分化、增殖、分裂和运动，（4）蛋白质通过膜的易位，（5）细胞内囊泡的运输，以及囊泡介导的分泌和摄取，通过gtp酶控制囊泡外壳组装。oncogene侧是诱发癌症的基因。

gαs pull-downactivation assay kit

cat.# 80801

introduction

a.background

a structurally diverse repertoireof ligands, from photons to large peptides, activates gprotein-coupled receptors (gpcrs) to elicit their physiologicalfunctions. ligand-bound gpcrs, in turn, function as guaninenucleotide exchange factors catalyzing the exchange of gdp bound onthe gα subunit with gtp in the presence of gβγ, causing thedissociation of the gα subunit from the gβγ dimer to form twofunctional units (gα and gβγ). both gα and gβγ subunits signal tovarious cellular signaling pathways. based on the sequence andfunctional homologies, g proteins are grouped into four families:gs, gi, gq, and g12.

gαs familyrelays signals from many gpcrs to regulate various biologicalfunctions such as the stimulation of adenylyl cyclases. there wereno direct methods to measure the activation of gαs proteinsby receptors (until this assay kit). most reports used onedownstream pathway, the increase of camp, as a readout.

gαs activationassay kit is based on the monoclonal antibody specificallyrecognizing the active gtp-bound gαs proteins.this monoclonal antibody has much lower affinity towards theinactive gαs proteins.therefore, after activation by receptor signals, active gtp-boundgαs proteinscould be immunoprecipitated by this monoclonal antibody and further by western blot with another anti-gαs antibody.

b.assay principle

the gαs activationassay kit uses configuration-specific anti-gαs-gtpmouse monoclonal antibody to measure gαs-gtplevels in cell extracts or in vitro gtpγs loading gαs activationassays. anti-gαs-gtpmouse monoclonal antibody is first incubated with cell lysatescontaining gαs-gtp.next, the gtp-bound gαs ispulled down by protein a/g agarose. finally, the precipitatedgαs-gtpis detected through immunoblot analysis using anti-gαs mousemonoclonal antibody.

c.kit components

1. anti-gαs-gtpmouse monoclonal antibody (cat. # 26906): one vial – 35 µl (1mg/ml) in pbs, ph 7.4, containing 50% glycerol. this antibodyspecifically recognizes gαs-gtpfrom all vertebrates.

2. protein a/g agarose (cat. #30301): one vial – 600 µl of 50% slurry.

3. 5x assay/lysis buffer (cat. #30302): one bottle – 30 ml of 250 mm tris-hcl, ph 8, 750mm nacl, 50mm mgcl2, 5 mm edta, 5% triton x-100.

4. anti-gαs mousemonoclonal antibody (cat. # 26006): one vial – 50 µl (1mg/ml) inpbs, ph 7.4, contained 50% glycerol.

5. 100x gtpγs (cat. # 30303): onevial – 50 µl at 10 mm, use 5 µl of gtpγs for  gtp-labeling of0.5 ml of cell lysate.

6. 100x gdp (cat. # 30304): onevial – 50 µl at 100 mm, use 5 µl of gdp for gdp-labeling of 0.5 mlof cell lysate.

7. hrp-goat anti-rabbit igg (cat.#29002): 50 µl (0.4 mg/ml) in pbs, ph 7.4, contained 50%glycerol.

d.materials needed but not supplied

1. stimulated and non-stimulatedcell lysates

2. protease inhibitors

3. 4 °c tube rocker orshaker

4. 0.5 m edta at ph8.0

5. 1.0 m mgcl2

6. 2x reducing sds-page samplebuffer

7. electrophoresis andimmunoblotting systems

8. immunoblotting wash buffersuch as tbst (10 mm tris-hcl, ph 7.4, 0.15 m nacl, 0.05% tween-20)

9. immunoblotting blocking buffer(tbst containing 5% non-fat dry milk or 3% bsa)

10. ecl detectionreagents

e.example results

the following figure demonstratesexample results seen with the gαs activationassay kit. for reference only.

gαs activationassay.purifiedgαs proteinswere loaded as a control (lanes 1) or immunoprecipitated aftertreated with gdp (lane 2) or gtpγs (lane 3). immunoprecipitationwas done with the anti-gαs-gtpmonoclonal antibody (cat. # 26906). immunoblot was with ananti-gαs monoclonalantibody (cat. # 26006).

assayprocedure

a.reagent preparation

1x assay/lysis buffer: mixthe 5x stock (cat. # 30301) briefly and dilute to 1x in deionizedwater. just prior to usage, add protease inhibitors such as 1 mmpmsf, 10 µg/ml leupeptin, or 10 µg/ml aprotinin.

b.sample preparationadherent cells

1. culture cells (one 10-cmplate, ~107 cells)to approximately 80-90% confluence. stimulate the cells withactivator or inhibitor as desired.

2. aspirate the culture media andwash twice with ice-cold pbs.

3. completely remove the finalpbs wash and add ice-cold 1x assay/lysis buffer (see reagentpreparation) to the cells (0.5-1 ml per 10 cm tissue cultureplate).

4. place the culture plates onice for 10-20 minutes.

5. detach the cells from theplates by scraping with a cell scraper.

6. transfer the lysates toappropriate size tubes and place on ice.

7. if nuclear lysis occurs, thecell lysates may become viscous and difficult to pipette. if thisoccurs, lysates can be passed through a 27½-gauge syringe needle3-4 times to shear the genomic dna.

8. clear the lysates bycentrifuging at 12,000 x g and 4°c for 10 minutes.

9. collect the supernatant andstore the sample (~1-2 mg of total protein) on ice for immediateuse, or snap freeze and store at -70°c for future use.

adherent cells

1. culture cells and stimulatewith activator or inhibitor as desired.

2. perform a cell count and thenpellet the cells through centrifugation.

3. aspirate the culture media andwash twice with ice-cold pbs.

4. completely remove the finalpbs wash and add ice-cold 1x assay/lysis buffer (see reagentpreparation) to the cell pellet (0.5-1 ml per 107 cells).

5. lyse the cells by repeatedpipetting.

6. transfer the lysates toappropriate size tubes and place them on ice.

9. collect the supernatant andstore sample on ice for immediate use, or snap freeze and store at-70°c for future use.

c.in vitro gtpγs/gdp protein for positive and negativecontrols

note: in vivo stimulation of cells willactivate approximately 10% of the available gαs,whereas in vitro gtpγs protein loading will activate nearly 90% ofgαs.

1. aliquot 0.5 ml of cell extract(or 1 µg of purified gαs protein)into two microcentrifuge tubes.

2. to each tube, add 20 µl of 0.5m edta (final concentration of 20 mm).

3. positive control: add 5 µl of100 x gtpγs (cat. # 30302) to the 1st tube

4. negative control: add 5 µl of100 x gdp (cat. # 30304) to the 2nd tube.

5. incubate both tubes at 30°cfor 30 minutes with agitation.

6. stop loading by placing thetubes on ice and adding 32.5 µl of 1 m mgcl2 (finalconcentration of 60 mm).

d.affinity precipitation of activated g protein

1. aliquot 0.5-1 ml of celllysates (about 1 mg of total cellular protein) to a microcentrifugetube.

2. adjust the volume to 1 ml with1x assay/lysis buffer (see reagent preparation).

3. add 1 µl anti-gαs-gtpantibody (cat. # 26906).

4. prepare the protein a/gagarose bead slurry (cat. # 30301) by resuspending throughvertexing or titrating.

5. add 20 µl ofresuspended bead slurry to above tube.

6. incubate the tube at 4°c for 1hour with gentle agitation.

7. pellet the beads throughcentrifugation at 5,000 x g for 1 min.

8. aspirate and discard thesupernatant (making sure not to disturb or remove the beadpellet.

9. wash the beads 3 times with0.5 ml of 1x assay/lysis buffer, centrifuging and aspirating eachtime.

10. after the third wash, pelletthe beads through centrifugation and carefully remove all thesupernatant.

11. resuspend the bead pellet in20 µl of 2x reducing sds- page sample buffer.

12. boil the sample for 5minutes.

13. centrifuge it at 5,000 x gfor 10 seconds.

e.western blot analysis

1. load 15 µl/well of pull-downsupernatant to a polyacrylamide gel (17%). it is recommended toinclude a pre-stained mw standard (as an indicator of a successfultransfer in step 3 below).

2. perform sds-page following themanufacturer’s instructions.

3. transfer the gel proteins to apvdf or nitrocellulose membrane following the manufacturer’sinstructions.

note: steps 4-11 are at room temperaturewith agitation

4. following electroblotting,immerse the pvdf membrane in **** methanol for 15 seconds, and thenallow it to dry at room temperature for 5 minutes.

note: if nitrocellulose is used insteadof pvdf, step 4 should be skipped.

5. block the membrane with 5%non-fat dry milk or 3% bsa in tbst for 1 he at room temperaturewith constant agitation.

6. wash the blotted membranethree times with tbst, 5 minutes each time.

7. incubate the membrane withanti-gαs mousemonoclonal antibody (cat. # 26006), which has been freshly diluted1: 50~500 (depending on the amount of gαs proteinsin your sample) in 5% non-fat dry milk or 3% bsa in tbst, for 1-2her at room temperature with constant agitation or at 4°covernight.

8. wash the blotted membranethree times with tbst, 5 minutes each time.

9. incubate the membrane with asecondary antibody (cat. # 29002), which has been freshly diluted1: 1000 in 5% non-fat dry milk or 3% bsa in tbst, for 1 he at roomtemperature with constant agitation.

10. wash the blotted membranethree times with tbst, 5 minutes each time.

11. use the detection method ofyour choice such as ecl.