Bio-Plex Multiplex Phosphoprotein Assay Protocol Outline For full instructions see BioPlex Binder or the protocol online

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

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

(! Remo'e the plate sealer and filter #ash -< #ith 00 l Bio-Plex phosphoprotein wash buffer 3! Resuspend the beads in each #ell #ith )+ l of Bio-Plex bea5 resuspension buffer. 0! 1o'er the #ells #ith plastic adhesi'e plate sealer& and thoroughly blot the bottom of the plate. ! 2ha,e the plate at +.+)) rp- 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#

Bio-Plex >ultiplex Phosphoprotein%ssay "ilution ?or,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 () l7well l of bead *+0x! stoc, solutionB#ell AAAAAAAA x l C AAAAAAA of bead *+0x! stoc, solution *assay module!

AAAAAAAA x 43 l C AAAAAA of Bio-Plex Phosphoprotein 8ash Buffer *phosphoprotein reagent ,it! AAAAAAAA x +0 l C AAAAAA total 'olume

Detection Antibo5y Dilution 2( l7well l of detection antibody *2(x! stoc, solutionB#ell AAAAAAAA x l C AAAAAAA of detection %b *)+x! stoc, solution *assay module!

AAAAAAAA x )4 l C AAAAAA of Bio-Plex Detection Ab Diluent A *phosphoprotein reagent ,it! AAAAAAAA x )+ l C AAAAAA total 'olume Strepta,i5in-PE Dilution () l7well 0.+ l of strepta'idin-P8 * 00x! stoc, solutionB#ell AAAAAAAA x 0.+ l C AAAAAAA of strepta'idin-P8 * 00x! stoc, solution *phosphoprotein reagent ,it! AAAAAAAA x 43.+ l C AAAAAA of Bio-Plex Phosphoprotein wash buffer *phosphoprotein reagent ,it! AAAAAAAA x +0 l C AAAAAA total 'olume