ing )0u/ of this 0 dilution per sample to spot duplicate #ells. The beginning of the detection antibody step is a good time! PREPARE BEADS ! "etermine the total number of #ells that #ill be re$uired. before each 'acuum filtration step to pre'ent damaging filter plate. Turn on the Bio-Plex instrument at least 40 minutes prior to plate reading.ing Bead solution at medium speed for -0 sec and add () µ l to each #ell. b! Protect the beads from light. a! %s a precaution& add 2 extra #ells for e'ery ( #ells actually re$uired. c! .com Bring all buffers and diluents to room temp prior to use. 6! Filter #ash 2* #ith +)) µ l Bio-Plex phosphoprotein wash buffer. solution in Bio-Plex phosphoprotein wash buffer. +! %dd Beads. 4! 5acuum filter. /ysates should be diluted to target concentration in lysis buffer& then diluted 0 #ith Bio-Plex assay buffer% Recommend ma. ASSA$ ! 1o'er all unneeded #ells #ith plastic adhesi'e plate sealer. a! Vortex for ( sec and immediately add () µ l to the appropriate #ells. . b! Remember to also include the appropriate blan. a! Vortex for ( sec and immediately add () µ l to the appropriate #ells.s *lysis buffer! 3! %dd samples. )! 2et the 'acuum pressure to 2 inches &' using a standard flat bottom 34-#ell plate. -! Pre-#et the filter plate #ith 00 µl of Bio-Plex phosphoprotein wash buffer% 4! 5acuum filter.. a! Vortex the #or. Blot the bottom of plate after this and e'ery 'acuum filtration. 1hec.ing dilution of the anti-phosphoprotein bead *+0x! stoc.Bio-Plex Multiplex Phosphoprotein Assay Protocol Outline For full instructions see BioPlex Binder or the protocol online at BioRad. )! Vortex the anti-phosphoprotein bead *+0x! stoc. (! %dd positi'e and negati'e controls. -! Prepare a +0 fold #or.eep all bead solutions on ice #hen not in use PREPARE O!"RO# A!D SAMP#E E## #$SA"ES ! Tha# positi'e and negati'e control and sample cell lysates on ice. a! "ocument all 'olume calculations. solution at medium speed for -0 sec.

4! 1o'er the #ells #ith plastic adhesi'e plate sealer& and blot the bottom of the plate.ing. a! %s a precaution& add 2 extra #ells for e'ery ( #ells actually re$uired. solution. a! "ocument all 'olume calculations. -! Prepare a 00 fold #or.te-perature #ith sha. a! 2lo#ly ramp up to +.eep all antibody solutions on ice #hen not in use.ing dilution of the detection antibody *)+x! stoc. 4! Remo'e the plate sealer and filter #ash -< #ith 00 µl Bio-Plex phosphoprotein wash buffer% +! 3ently .0! 1o'er the #ells #ith plastic adhesi'e plate sealer& and blot the bottom of the plate.ing dilution of the 2trepta'idin-P8 * 00x! stoc. )! Vi'orously .te-p #ith sha.ortex the detection antibody and add 2( ul to each #ell.+)) rp.+)) rp. b! Protect the solution from light.ing dilution and add () µ l to each #ell.te-perature #ith sha. a! 2lo#ly ramp up to +.eep on ice #hen not in use 4! Remo'e the plate sealer and filter #ash -< #ith 00 µl Bio-Plex phosphoprotein wash buffer. solution in Bio-Plex phosphoprotein 5etection antibo5y 5iluent.e at high speed for /) sec b! Reduce speed to /)) rpm for the remainder of incubation PREPARE DE"E "0O! A!"0BOD$ 1D2R0!3 PRE EED0!3 0! 2BA"0O! A!D +) M0! PR0OR "O 2SE4 ! "etermine the total number of #ell that #ill be re$uired. a! %s a precaution& add 2 extra #ells for e'ery ( #ells actually re$uired. ! 1o'er #ith foil and incubate o.erni'ht at roo.e at high speed for /) sec b! Reduce speed to /)) rpm for the remainder of incubation PREPARE S"REP"AV0D0!-PE 1D2R0!3 PRE EED0!3 0! 2BA"0O! A!D +) M0! PR0OR "O 2SE4 ! "etermine the total number of #ells that #ill be re$uired.RT8< the multiplex detection antibody *)+x! stoc. solution in in Bio-Plex phosphoprotein wash buffer. 6! 1o'er #ith foil and incubate for +) -in at roo.ing. a! 2lo#ly ramp up to +.and sha.ortex the 2trepta'idin-P8 * 00x! stoc.ing. solution.and sha. -! Prepare a )+ fold #or.+)) rp.e at high speed for /) sec b! Reduce speed to /)) rpm for the remainder of incubation . c! . 6! 1o'er #ith foil and incubate for /) -in at roo. +! Vortex the strepta'idin-P8 #or. 4! 1o'er the #ells #ith plastic adhesi'e plate sealer& and blot the bottom of the plate. a! "ocument all 'olume calculations. b! Protect the antibody from light. )! 789T/: 5.and sha. c! .

+)) rp.(! Remo'e the plate sealer and filter #ash -< #ith 00 µl Bio-Plex phosphoprotein wash buffer 3! Resuspend the beads in each #ell #ith )+ µl of Bio-Plex bea5 resuspension buffer. ! 2ha. 0! 1o'er the #ells #ith plastic adhesi'e plate sealer& and thoroughly blot the bottom of the plate.for /) sec = slo#ly ramp up to this high speed )! Remo'e the sealer and read the plate. !O"E6 Set the Bio-Plex Rea5er to rea5 only 2( bea5s7re'ion in a .olu-e of () u# .e the plate at +.

Bio-Plex >ultiplex Phosphoprotein%ssay "ilution ?or. solution *assay module! AAAAAAAA x 43 µl C AAAAAA of Bio-Plex Phosphoprotein 8ash Buffer *phosphoprotein reagent .it! AAAAAAAA x +0 µl C AAAAAA total 'olume .sheet @ of re$uired #ells AAAAAA @ of extra #ells AAAAAA *) #ells for e'ery ( re$uired #ells! AAAAAAA total number of #ells for dilution calculations Bea5 Dilution () µ l7well µl of bead *+0x! stoc. solutionB#ell AAAAAAAA x µl C AAAAAAA of bead *+0x! stoc.

Detection Antibo5y Dilution 2( µ l7well µl of detection antibody *2(x! stoc. solution *assay module! AAAAAAAA x )4 µl C AAAAAA of Bio-Plex Detection Ab Diluent A *phosphoprotein reagent . solution *phosphoprotein reagent . solutionB#ell AAAAAAAA x 0.+ µl C AAAAAA of Bio-Plex Phosphoprotein wash buffer *phosphoprotein reagent .+ µl C AAAAAAA of strepta'idin-P8 * 00x! stoc.i5in-PE Dilution () µ l7well 0.+ µl of strepta'idin-P8 * 00x! stoc.it! AAAAAAAA x 43.it! AAAAAAAA x )+ µl C AAAAAA total 'olume Strepta.it! AAAAAAAA x +0 µl C AAAAAA total 'olume . solutionB#ell AAAAAAAA x µl C AAAAAAA of detection %b *)+x! stoc.