Gαi 活性检测试剂盒丨免疫检测试剂盒

gαi pull-downactivation assay kit

cat. #83001

introduction

a.background

a structurally diverse repertoire of ligands, from photons tolarge peptides, activates g protein-coupled receptors (gpcrs) toelicit their physiological functions. ligand-bound gpcrs, in turn,function as guanine nucleotide exchange factors catalyzing theexchange of gdp bound on the gα subunit with gtp in the presence ofgβγ, causing the dissociation of the gα subunit from the gβγ dimerto form two functional units (gα and gβγ). both gα and gβγ subunitssignal to various cellular signaling pathways. based on thesequence and functional homologies, g proteins are grouped intofour families: gs, gi, gq, and g12.

gαi familyis the largest family of g proteins. they relay signals from manygpcrs to regulate various biological functions. there were nodirect methods to measure the activation of gαi proteinsby receptors (until this assay kit). most reports used one of thedownstream pathways, i.e. the inhibition of adenylyl cyclases, as areadout. alternatively, sensitivity to pertussis toxin (ptx) wasused as an indicator of possible gαi proteinsinvolved in a signaling pathway.

b. assayprinciple

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

c. kitcomponents

1. anti-gαi-gtpmouse monoclonal antibody (cat. # 26901): one vial – 35 µl (1mg/ml) in pbs, ph 7.4, containing 50% glycerol. this antibodyspecifically recognizes gαi-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 of250 mm tris-hcl, ph 8, 750mm nacl, 50 mm mgcl2, 5 mm edta, 5%triton x-100.

4. anti-gαi mousemonoclonal antibody (cat. # 26003): one vial – 50 µl (1mg/ml) inpbs, ph 7.4, contained 50% glycerol.

5. 100x gtpγs (cat. # 30303): one vial – 50 µl at 10 mm, use 5µl of gtpγs for  gtp-labeling of 0.5 ml of cell lysate.

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

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

d.materials needed but not supplied

1. stimulated and non-stimulated cell lysates

2. protease inhibitors

3. 4 °c tube rocker or shaker

4. 0.5 m edta at ph 8.0

5. 1.0 m mgcl2

6. 2x reducing sds-page sample buffer

7. electrophoresis and immunoblotting systems

8. immunoblotting wash buffer such as tbst (10 mm tris-hcl, ph7.4, 0.15 m nacl, 0.05%  tween-20)

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

10. ecl detection reagents

e.example results

the following figure demonstrates example results seen with thegαi activationassay kit. for reference only.

gαi activationassay. 

a. cho cells were transfected with wild-type gαi1 (lanes1 and 2) or constitutively active gαi1-q204l(lane 3). cell lysates were treated with gdp (lane 1) or gtpγs(lane 3). lysates were then incubated with ananti-gαi-gtpmonoclonal antibody (cat. # 26901) (top panel). the precipitatedgαi-gtpwas immunoblotted with an anti-gαi monoclonalantibody (cat. # 26003). the bottom panel shows the western blotwith anti-gαi monoclonalantibody (cat. # 26003) of the cell lysates. b. hek293 cells stablyexpressing human m2 machr were treated with (lane 2) or without(lane 1) carbachol. cell lysates were then incubated with ananti-active gαi monoclonalantibody (cat. no. 26901) (top panel). the precipitatedgαi-gtpwas immunoblotted with an anti-gαi rabbitpolyclonal antibody (cat. # 21006). the bottom panel shows thewestern blot with anti-tubulin of the cell lysates.

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-cm plate, ~107 cells)to approximately 80-90% confluence. stimulate the cells withactivator or inhibitor as desired.

2. aspirate the culture media and wash twice with ice-coldpbs.

3. completely remove the final pbs wash and add ice-cold 1xassay/lysis buffer (see reagent preparation) to the cells (0.5-1 mlper 10 cm tissue culture plate).

4. place the culture plates on ice for 10-20 minutes.

5. detach the cells from the plates by scraping with a cellscraper.

6. transfer the lysates to appropriate size tubes and place onice.

7. if nuclear lysis occurs, the cell lysates may become viscousand difficult to pipette. if this occurs, lysates can be passedthrough a 27½-gauge syringe needle 3-4 times to shear the genomicdna.

8. clear the lysates by centrifuging at 12,000 x g and 4°c for10 minutes.

9. collect the supernatant and store the sample (~1-2 mg oftotal protein) on ice for immediate use, or snap freeze and storeat -70°c for future use.

adherent cells

1. culture cells and stimulate with activator or inhibitor asdesired.

2. perform a cell count and then pellet the cells throughcentrifugation.

3. aspirate the culture media and wash twice with ice-coldpbs.

4. completely remove the final pbs wash and add ice-cold 1xassay/lysis buffer (see reagent preparation) to the cell pellet(0.5-1 ml per 107 cells).

5. lyse the cells by repeated pipetting.

6. transfer the lysates to appropriate size tubes and place themon ice.

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

c. invitro gtpγs/gdp protein for positive and negativecontrols

note: in vivo stimulation ofcells will activate approximately 10% of the availablegαi,whereas in vitro gtpγs protein loading will activate nearly 90% ofgαi.

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

2. to each tube, add 20 µl of 0.5 m edta (final concentration of20 mm).

3. positive control: add 5 µl of 100 x gtpγs (cat. # 30302) tothe 1st tube

4. negative control: add 5 µl of 100 x gdp (cat. # 30304) to the2nd tube.

5. incubate both tubes at 30°c for 30 minutes withagitation.

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

d.affinity precipitation of activated g protein

1. aliquot 0.5-1 ml of cell lysates (about 1 mg of totalcellular protein) to a microcentrifuge tube.

2. adjust the volume to 1 ml with 1x assay/lysis buffer (seereagent preparation).

3. add 1 µl anti-gαi-gtpantibody (cat. # 26901).

4. prepare the protein a/g agarose bead slurry (cat. # 30301) byresuspending through vertexing or titrating.

5. add 20 µl of resuspended bead slurry to abovetube.

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

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

8. aspirate and discard the supernatant (making sure not todisturb or remove the bead pellet.

9. wash the beads 3 times with 0.5 ml of 1x assay/lysis buffer,centrifuging and aspirating each time.

10. after the third wash, pellet the beads throughcentrifugation and carefully remove all the supernatant.

11. resuspend the bead pellet in 20 µl of 2x reducing sds- pagesample buffer.

12. boil the sample for 5 minutes.

13. centrifuge it at 5,000 x g for 10 seconds.

e.western blot analysis

1. load 15 µl/well of pull-down supernatant to a polyacrylamidegel (17%). it is recommended to include a pre-stained mw standard(as an indicator of a successful transfer in step 3 below).

2. perform sds-page following the manufacturer’sinstructions.

3. transfer the gel proteins to a pvdf or nitrocellulosemembrane following the manufacturer’s instructions.

note: steps 4-11 are at roomtemperature with agitation

4. following electroblotting, immerse the pvdf membrane in methanol for 15 seconds, and then allow it to dry at roomtemperature for 5 minutes.

note: if nitrocellulose isused instead of pvdf, step 4 should be skipped.

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

6. wash the blotted membrane three times with tbst, 5 minuteseach time.

7. incubate the membrane with anti-gαi mousemonoclonal antibody (cat. # 26003), which has been freshly diluted1: 50~500 (depending on the amount of gαi 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 membrane three times with tbst, 5 minuteseach time.

9. incubate the membrane with a secondary antibody (cat. #29002), which has been freshly diluted 1: 1000 in 5% non-fat drymilk or 3% bsa in tbst, for 1 he at room temperature with constantagitation.

10. wash the blotted membrane three times with tbst, 5 minuteseach time.

11. use the detection method of your choice such as ecl.