From ELISA to Epigenetics, a FASTER,

EASIER way to run complex Assays

Maxine Santoro Ph. D.Field Application Scientist

© 2011 Perkin Elmer

Alpha Technology

University of Iowa, October 3, 2012

Agenda

Alpha Technology- How it works/ why it is different

Assay Examples- What kind of assays you can run

ELISA- Comparison of ELISA vs. AlphaLISA assaysPhosphoprotein Detection- Comparison of

Western Blot vs. Alpha SureFireProtein:Protein Interactions- Kinase: protein interaction

assaysEpigenetics Assays

How will Alpha Technology help your lab?

Principle of the Alpha Technology

Bead based assay-Assay reagents link to beads and bring beads into close proximity allowing a signal to be generated

Time resolved/low backgroundSinglet oxygen can travel 200nm before decayLow energy to high energy Large amplification due to chemical reaction in Acceptor beads

Amplified Luminescence Proximity Homogeneous Assay

AlphaScreen® Technology Summary

• Background:– Developed by Dade Behring, Inc., in 1994 as LOCI– PerkinElmer has rights to technology for research only

• Latex Bead-based System– Donor & Acceptor beads– ~250 nm diameter, each with unique chemistries

• Stable Colloidal Suspension– stay suspended (>12 hrs)– will not clog pipettes– can be centrifuged

• Coated with a Dextran Polymer Hydrogel

– prevents non-specific interaction & aggregation– contains reactive aldehyde groups for conjugation

Biological Interactions Measured with Alpha Technology

Biological Interactions Measured with Alpha Technology

AlphaLISA immunoassays: assay formats

So why do alpha?

Sandwich Assay

Sign

al

Endogenous analyte

Streptavidin-coated Donor bead with biotinylated antibody

Analyte

Antibody coupled to Acceptor bead

More than 1 Ab to AnalyteCompetition Assay

-12 -11 -10 -9 -8 -7

Sign

al

Endogenous analyte

Competing analyte

Streptavidin-coated Donor bead with biotinylated antibody

BiotinylatedAnalyte

Antibody coupled to Acceptor bead

Few Abs to Analyte or Small Analyte

ELISA

AlphaLISA

AlphaLISA comparison to ELISA

Easier protocol

AlphaLISA compare to traditional ELISA?

AlphaLISA is bead based, technically different…but similar in functionality (sandwich assay in solution)

No wash vs wash (time consuming)Scalable (from 96-well to 1536-well)Very wide dynamic range

When to consider utilizing AlphaLISA?Targeted analyte (kits)Desire for a simple protocol with no

dilutions and wash stepsWorking with low affinity interactions

that can be disrupted by washingAutomation, miniaturization & higher

throughput needed

Add assay buffer, matrix solution, standard(or sample); Add antibody detection solution

ReadIncubate 1 hour shaking then wash well

Add enzyme

Incubate 30 minutes shaking then wash well

Add substrateIncubate 30 minutes shaking

ELISA (4-8 hours)

60 30

Add assay buffer, matrix solution, standard (or sample); Add biotin antibody and AlphaLISA acceptor beads

ReadIncubate 30-60 minutes (Room Temperature)

Add donor beads

Incubate 30-60 minutes (Room Temperature)

AlphaLISA (2 hours)

30-60 30-60

ELISA vs AlphaLISA

…very good correlation between the two assays

0 50 100 150 200 2500

100

200

300

400

r2= 0.9970

AlphaLISA

ELI

SA

0 100 200 300 400 500 600 700 8000

200

400

600

800

1,000

r2= 0.9823

AlphaLISA

ELI

SA

AlphaLISA versus ELISA …

IL 1-BetaIL- 8

IL8 and IL1β (UMass Medical Center, US)Cell culture supernatants

AlphaLISA versus ELISA …

R2 = 0.962

Insulin data correlation betweenMercordia ELISA and AlphaLISA kit

0

10

20

30

40

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Mercodia insulin (ng/mL)

Alp

haLI

SAin

sulin

(ng/

mL)

…very good correlation between the two assays

Insulin kit (Roche, New Jersey)18 plasma samples tested (diluted 10

times in AlphaLISA buffer)

Page 12

Summary of Alpha Benefits

* LOCI = AlphaLISA

> 10 fold> 2 fold> 5 fold

Poulsen and Jensen: JBS 12(2) 240-247, 2007

AlphaLISA – Effect of serum dilution

)

Experiment

Dilution[expected

insulin] n=1 n=2 n=3 Average % of expected 1 100 114 124 112 117 1171/2 50 48 53 52 51 1021/3 33.3 40 34 36 37 1121/4 25 30 23 29 27 109 0 0 1 1 1 1 n/a

Linearity (insulin content in uIU/ml)

Conclusion: AlphaLISA samples can be diluted with serum (analyte depleted) and the results correspond to the expected dilution a 1:2 dilution yields half the signal

Last 4 slides show ELISA and AlphaLISA give expected results-- both good technologies What makes Alpha better than ELISA assays?

What makes Alpha better than ELISA assays?

A human serum sample spiked with 100 µIU/mL was diluted in matrix solution

(serum with insulin removed)

AlphaLISA – Assay Precision

Intra-assay variation (insulin content in uIU/ml)replicate

[spiked insulin] 1 2 3 4 5 6 Average %CV 85 74 76 84 85 86 81 81 648 41 44 46 52 51 48 47 927 23 23 24 29 29 26 26 1118 15 17 17 21 20 20 18 123 3 5 7 5 4 5 5 23

Inter-assay variation (insulin content in uIU/ml)Experiment

[spiked insulin] n=1 n=2 n=3 n=4 n=5 n=6 Average %CV85 90 84 77 98 96 82 88 948 49 51 43 54 50 58 51 1027 26 28 24 26 26 27 26 618 17 20 17 19 17 18 18 73 4 5 6 4 4 4 4 17

Conclusion: Small variation higher concentrations, At lower concentrations nearing LDL (1.5 uIU) variation increases

-same plate

-different experiments/days

Assay precision-5 samples spiked with various concentrations of insulin 6 replicates

Typical standard curve analysis

S/B = maximum signali i i l

 

100

1,000

10,000

100,000

1,000,000

-13 -12 -11 -10 -9 -8 -7-

LDL : 10.9 pg/mL

Log [rat C-Peptide-1] (g/mL)

Alp

haLI

SA S

igna

l (co

unts

)

Maximum signal

Minimum signal

LDLAverage bkg (n=12) + 3SD

Dynamic

range

Lower Detection Limit- lowest reading where obtain significant difference from background

Dynamic range- Range from lowest detection limit to highest level of detection

HDLAverage (n=12) - 3SD

Standard Curves of TNF ELISA vs. AlphaLISAComparison of sensitivity and range of detection

0.1 1 10 100 1000 10000 100000 1000000100

1000

10000

100000

1000000

AlphaLISA

ELISA

0

1

2

3

4

[TNF-] (pg/mL)

Alp

haSc

reen

(Cou

nts)

ELISA (OD

)

Dynamic Range

15 pg/ml ELISA vs. 1.5 pg/ml AlphaLISALower limit of detection

2 logs for ELISA vs. 4 logs for AlphaLISA

mIL6 assay on collection of Samples-Standard Curve

M IL-6 Graph on Enspire

IL6 Alpha Calculated in 50 ul Assay

sample number calc values1

2

3

4

5 2.0106136 39.630637

8

9

10

11

12 85.6307113

14 37.5225415 4.57573116 84.6006917

18 28.6859519 0.795121620 60.26664

43.92521

103.3413

41.07347

102.0545

28.93566

71.13919

Enspire data

Not visible in ELISA

50 ul Standard Curve

Bottom of curve=680R2=.97

Goes to .093?

Standard Curve for 10 ul Assay

LDL=.56 pg/mlBottom of curve=295R2=.92

LDL goes to .439

Summary of IL6 AlphaLISA-Assays are very easy to do -Very low data variability -commensurate with pipetting s-Very sensitive from 1.2 pg/ml detection-Need to make certain that matrix for sample is the sameas for samples-Easy to miniaturize

EPO Determination

In AlphaLISA buffer:LDL: 1.0 mIU/mlDynamic range: 1 – 30,000 pg/mlIn Analyte-depleted serum:LDL: 5.8 mIU/mlDynamic range: 5.8 – 30,000 pg/ml

AlphaLISA Summary

AlphaLISA is:

EASY: all ELISA assays can be easily converted to the AlphaLISA platform

FLEXIBLE: AlphaLISA can detect a broad range of analytes in complex biological matrices

KEY advantages of AlphaLISA over ELISA are: Homogeneous (no wash steps)Highly sensitive with a Wide Dynamic RangeSimple (3 addition steps) and fast protocolScalable (96, 384 and 1536 formats) and easily automatableValidated on PerkinElmer InstrumentsLess hands on timeCost-effective

What Else Can you do with Alpha?

√ AlphaLISASignificantly less steps, less time (< 2 hours)Superior dynamic range, great sensitivityLow sample volume- 5 l

SureFire PhosphoProtein DetectionComparable results to your Western-EasierElimination of imaging/ problems with uneven blots

Protein: Protein Interaction AssaysAbility to use large proteins- unlike Fluorescent polarization or FRETAbility to measure low affinity mM interactions

Epigentics AssaysAlpha and TR-FRET assaysMethylation and Acetylation assays

AlphaLISA vs. SureFire Differences

Immunoassay ELISA assay--Streptavidin Donor beads

--Biotin Ab--analyte--Ab Direct conjugation to Acceptor beads

Immunoassays ELISA ASSAY CONFIGURATIONS

Immunoassay ELISA assay--Streptavidin Donor beads--biotin AB----analyte----Ab linked to Protein A Acceptor beads

520-620 nm

615 nm

AlphaLISA Configuration

SureFire Configuration

Biotinylated Ab can not bind to Protein A

Use biotinylated mouse IgG1 antibody to avoid interference with Protein A.

Current AlphaScreen® SureFire® Kits

AlphaScreen® SureFire® Cellular Kinase Assays

Add cells

Adherent cells: Incubate overnight

Non-adherent cells:Assay immediately

Add Inhibitors Add Stimulant

Adherent cells: Add concentrated stock direct to wells

Non-adherent cells:Add concentrated stock direct to wells

Adherent cells: Remove media first and lyse with 1X Lysis buffer orAdd 5X Lysis buffer direct to wells

Non-adherent cells:Add 5X Lysis buffer direct to wells

Adherent cells: Remove media first or Add concentrated stock direct to wells

Non-adherent cells:Add concentrated stock direct to wells

Lyse cells

General Protocol – cell culture

General Protocol

Culture plate

Assay plate

Assay Target 2 reagents

Assay Target 1 reagents

Assay Target 3 reagents

lysate

Culture plate

OR

Transfer lysate from the culture plate to a fresh microplate for assay

Assay Reagents

Assay Reagents

Perform assay in the culture plate

AlphaScreen® SureFire® Cellular Kinase Assays

« Transfer (2 plates) Assay »

« Single Plate Assay »« One plate Assay »« One-Well Assay »

Conclusion: Alpha SureFire produces equivalent results to Western Blot

SureFire vs. Western Blot Detection

Recombinant ERK1 Standard Curve

ERK Standard Curve

WesternSureFire

SureFire measures ERK-1 activation by agonist more accurately than Western Blot

Human Muscarinic Receptor2/ Agonists

Conclusion: Alpha SureFire produces equivalent results to Western Blot

GPCR Signaling to ERK• AlphaScreen Surefire pERK assay for

GPCR activation– Data indicates that most GPCRs can signal thru pERK – generic assay

• Large assay windows & S/B

Insulin receptor signaling pathway screenfor PI3K / AKT / mTOR

50000

100000

150000

200000

10-10 10-9 10-8 10-7 10-6 10-5

Log [Insulin] (M)

Rapamycin (mTOR)

Control insulin alone

U0126 (MEK)

Ly294002 (PI3K)

p70

S6K

Pho

spho

ryla

tion

(cou

nts)

AlphaScreen SureFire – Growth & Differentiation TargetsP70 S6K Phosphorylation (Thr389) in MCF-7 Cells

RasGTP

Raf

MEK

ERK

p70 S6K

P

P

P

P421/424

PI3K

PDK1

AKT

mTOR

P

P

PP389

L L

SureFire Selectivity Assay Example

Assay for PI3-kinase i hibit d ifi it

LY compound inhibits AKT pathwaybut not MEK pathway.

Conclusion: Able to run simultaneous assays using SureFire on one lysate

Kits

Comparison of Alpha SureFire and Western Blot Assays

Multiple Targets Limited bygel lanes

Analysis of 5 targetsin same pathway from one sample

Westerns SureFireSensitivity High High/Ultra kit

Assay Nature 20 steps 4-5 hrs. hands-on 8-24 hrs total

7 steps1-1. 5 hrs hands-on4-6 hours total

Throughput 10-20 lanes per gel Up to unlimitedsamples in parallel

Quantification Relative -based on band intensity

CV’s < 5%Normalize with totalAKT or GAPDH kits

Specificity 2 antibodies 2 antibodies

Anything Else you can do?

√ AlphaLISASignificantly less steps, less time (< 2 hours)Superior dynamic range, great sensitivityLow sample volume- 5 l

√ SureFire PhosphoProtein DetectionSignificantly less steps, less timeElimination of imaging/ problems with uneven blots

Protein: Protein Interaction AssaysAbility to use large proteins- unlike Fluorescent polarization or FRETAbility to measure low mM affinity interactions

Epigenetics AssaysAlpha and TR-FRET assaysMethylation and Acetylation assays

AlphaLISA ToolboxAlpha Donor Bead

. Prot

ein

A

Anti-

FLAG

Anti-

Mou

se

Anti-

Rabb

it

Stre

p -Tac

tin®

Ni c

hela

te

GSH

Stre

ptav

idin

Alp

haLI

SA A

ccep

tor B

ead

Anti-Chicken IgYAnti-cMycAnti-DigoxigeninAnti-FITCAnti-FLAGAnti-GFPAnti-Goat IgGAnti-GSTAnti-HisAnti-Human IgGAnti-MBPAnti-Mouse IgGAnti-Mouse IgMAnti-Rabbit IgGAnti-Rat IgGAnti-Sheep IgGAnti-V5GlutathioneNi chelateProtein AProtein GProtein LStrep -Tactin®

Streptavidin

You can use this pairThis pair might give high backgroundNot recommended

Alp

haLI

SA A

ccep

tor B

ead

--biotinylated P53 protein–--GST tagged HDM2 protein--anti-GST Acceptor Bead--Streptavidin Donor Bead-

P53-HDM2 Protein:Protein Assayusing tagged proteins

Protein:Protein Interaction Assay Examples

Tag detection Acceptor beads :

MEK1-ERK2 kinase activation model

In the unactive state, unphosphorylated MEK1 and ERK2 are tightly bound.Activated by phosphorylation, MEK1 phosphorylates and activates ERK2,resulting in ERK2 activation and complex dissociation.

Assay background:

Using Alpha -Ability to develop an assay measuring protein interaction and dissociation

Mathieu Arcand 2009-04-24 SBS MeetingBiochemistry, Vol. 49, No. 15, 2010

ERK- Kinase MW = 42kDMEK- Kinase MW = 50kD

41

MEK1-ERK2 activation model

In the unactive state, unphosphorylated MEK1 and ERK2 are tightly bound.Activated by phosphorylation, MEK1 phosphorylates and activates ERK2. This results in MEK1-ERK2 complex dissociation.

ERK1/2 MAP kinase pathway

42

MEK1 – ERK2 interaction assay01 nM3 nM10 nM30 nM100 nM

[MEK1]

0

50,000

100,000

150,000

200,000

250,000

10-10 10-9 10-8 10-7 10-60[ERK2 unactive] (M)

Alp

haLI

SA S

igna

l (co

unts

)

unactiveunactive

10-10 10-9 10-8 10-7 10-60[ERK2 unactive] (M)

activeunactive

10-10 10-9 10-8 10-7 10-60[ERK2 active] (M)

activeactive

MEK1-ERK2 interactions can be detected with an antibody-free AlphaLISA setup, and activation state of either protein greatly influences binding.

Emissionrecorded at520-620 nm

Gutathione-coated Donor Beads

Ni chelate conjugatedAlphaLISAAcceptor Beads

1O2

Excitation680 nm

43

MEK1 kinase assay on ERK2

0

50,000

100,000

150,000

200,000

01 nM3 nM10 nM30 nM100 nM

10-11 10-10 10-9 10-8 10-7 10-6

[MEK1]

0[ERK2] (M)

Alp

haSc

reen

Sig

nal (

coun

ts)

His-tagged active MEK1 was incubated with unphosphorylated GST-ERK2 in the presence of 10 µM ATP

Emissionrecorded at520-620 nm

Anti-mouse conjugatedAlphaScreen Acceptor Beads1O2

Excitation680 nm

Anti-pTpY ERK1/2antibody

Gutathione-coated Donor Beads

MEK1-ERK2 phosphorylation-interaction assay

44

1O2

Emission recorded at572 nm

Anti-mouse conjugatedAlphaScreen Acceptor Beads

1O2

Excitation680 nm

Anti-pTpY ERK1/2antibody

Gutathione-coated Donor Beads

Ni chelate conjugated AlphaLISAAcceptor Beads

Emission recorded at615 nm

0

10,000

20,000

30,000

40,000

50,000

10-9 10-6 10-3002,5005,0007,50010,00012,50015,00017,500

[ATP] (M)A

lpha

LISA

(615

nm c

ount

s)

Alp

haSc

reen

(572

nm c

ount

s)

Active His-tagged MEK1 was incubated with unphosphorylated GST-ERK2 in the presence of increasing ATP concentrations

We here provide the first direct evidence that ERK2 phosphorylation triggers itsdissociation from active MEK1.Both biomolecular events are intrinsically linked with interaction IC50 matchingERK2 phosphorylation EC50.

025,00050,00075,000

100,000125,000150,000175,000

10-9 10-6 10-30Alp

haLI

SA (6

15nm

cou

nts)

0

10,000

20,000

30,000

10-9 10-6 10-300

5,000

10,000

15,000

20,000

AlphaScreen

(572nm

counts)

Mechanism of Action of Kinase inhibitors

Sta

uros

pori

ne

IC50 = 0.3 nMIC50 = 15.7 µM

EC50 = 25.0 nMIC50 = 129 nM

InteractionPhosphorylation

+ATPNo ATP

unactiveunactive

activeunactive

ATP site competitor

025,00050,00075,000

100,000125,000150,000175,000

10-9 10-6 10-30Alp

haLI

SA (6

15nm

cou

nts)

0

10,000

20,000

30,000

10-9 10-6 10-300

5,000

10,000

15,000

20,000

AlphaScreen

(572nm

counts)

U01

26

IC50 = 1.6 nMIC50 = 10.4 µM

EC50 = 15.6 µMIC50 = 0.4 nM

Allosteric inhibitor

Conclusion: Different inhibitors display different profiles

Tightly bound Loss of binding

No ATP/ no phosphorylation

No ATP/ no phosphorylationBiphasic reduction in interaction assay

Loss of phosphorylation activity (staurosporine)Rescues binding at higher concentrations

Biphasic reduction in interaction assayDue to different binding affinities ofCompounds to MEK and ERK

Binding activity not rescuedBy allosteric inhibitor

Inhibitor [ ]

More uses…….

√ AlphaLISASignificantly less steps, less time (< 2 hours)Superior dynamic range, great sensitivityLow sample volume- 5 l

√ SureFire PhosphoProtein DetectionSignificantly less steps, less timeElimination of imaging/ problems with uneven blots

√ Protein: Protein Interaction AssaysAbility to use large proteins- unlike Fluorescent polarization or FRETAbility to measure low mM affinity interactions

Epigenetics AssaysAlpha and TR-FRET assaysMethylation and Acetylation assays

#1

#2

Epigenetics: The study of non-DNA sequence-related heredity

This is what determines what genes are expressed during development and involvedin cell differentiation and determines if a cells becomes a nerve cell, bone cell etc.

The Nucleosome

H2B

H2A

H3H4

Highest profile modificationsWriters: Acetylation (HAT) Methylation (HKMT and HRMT) Ubiquitination and Ubl (E1, E2, and E3) Phosphorylation (kinase)

Erasers : Deacetylation (HDACs and Sirtuins) Demethylation (demethylase)

Readers: Proteins that bind to the modified

histones and “act”

Our current products are focused on the WRITERS and ERASERs of Histone H3 and p53 with a tool box that allows the development of READERassays

© Richard Wheeler

In Vitro Enzymatic Assays

Toolbox for detecting Histone H3 & p53 modifications Over 30 enzyme assays developed

(including new assays for unmodified H3K9/K27 and H3R2me)

In vitro Toolbox

TR-FRETEmission665 nm

Excitation320 or 340 nm

Residual fluorescentemission 615 nm

FRET

biotin-peptide

bio SA-ULight TM

epigenetic markStreptavidin-ULight TM

Eu-labeledantibody

TR-FRET (LANCE) and AlphaLISA – Detection Assays Set-up

LANCE Ultra:Donor : W1024 - Eu chelate (conjugated to Ab)Acceptor : ULight (conjugated to Streptavidin)

Emission615nm

Anti-mark AlphaLISAAcceptor Bead

Excitation680 nm

Streptavidin-coatedAlpha Donor Bead

Modifiedpeptide

bio

TR-FRET set upSame assay using donor

and acceptor pairs

AlphaLISA set up Methylate or acetylatebiotinylated peptide

Methylation and Acetylation assays

epigenetic mark

150 ng/well EZH2 complex100 nM biotin-H3K27me0 peptide (H3 21-44)ATKAARKSAPATGGVKKPHRYRPGGK(Biotin)-OH3 µM SAM (Km,app)120 min reaction at 23°C (linearity verified) Detection with anti-H3K27me2-1 Acceptor beads<1% substrate turnover !!

Typical Signal-increase Assay: AlphaLISA EZH2 Methylation Assay

Optimized assay conditions:

0 30 60 90 120 150 1800

10,00020,00030,00040,00050,00060,00070,000

1501007550

[EZH2] (ng/well)

250

[SAM]: 100 µM

Time (min)

Alph

aLIS

A Si

gnal

(cou

nts)

0

10,000

20,000

30,000

40,000

50,000

-10 -9 -8 -7 -6 -5 -4 -3

Km app = 2.9 µM

Log [SAM] (M)

Alph

aLIS

A Si

gnal

(cou

nts)

-0

5,00010,00015,00020,00025,00030,00035,000

-6 -5 -4 -3 -2 -1-

IC50 = 420 M

Log [Sinefungin] (M)

Alph

aLIS

A Si

gnal

(cou

nts)

0 10 20 30 40 500

10,000

20,000

30,000

40,000No inhibitor

10 mM Sinefungin

Z' = 0.71S/B = 52

Well #Al

phaL

ISA

Sign

al (c

ount

s)

*

Substrate: recombinant Histone H3 (Active Motif) Uses anti-H3K4me1-2 Acceptor beads and biotinylated anti-Histone H3 (C-ter) antibodyHigh salt treatment required after the enzymatic assayUnique detection buffer optimized for bead dilution (different from Epigenetics Buffer 1)

AlphaLISA assay set-up with full-length histone H3

Validation of SET7/9 assay

Alpha

Histone H3 protein substrate

0 10 20 30 40 500

50,000

100,000

150,000

200,000

10 mM SinefunginZ' = 0.70S/B = 72

Total1 mM SinefunginZ' = 0.68S/B = 25

# wells

Alph

aLIS

A Si

gnal

(cou

nts)

0

50,000

100,000

150,000

200,000

-8 -7 -6 -5 -4 -3 -2 -1-

SinefunginIC50 = 47 µM

SAHIC50 = 175 µM

Log [Inhibitor] (M)

Alph

aLIS

A Si

gnal

(cou

nts)

In vitro Cell-based

H3R2me2

H3Thr3p

H3K4

H3K4me1-2

H3K4me2

H3K9

H3K9ac

H3K9me2

H3Ser10p

H3K27ac

H3K27me2

H3K27me3

H3K36me2

bio-Ab H3 (C-ter)

Detection buffer Epigenetics buffer 1

Buffer Set Cell-Histone (3)

DetectionHistone Mark

Hist

one

H3 D

etec

tion

Reag

ents

In Vitro:– techniques

Cellular:Kits for detection of endogenous histone H3 epigenetic

Epigenetics Portfolio Overview: In Vitro Assays

LANCE & AlphaLISATool box for developing in vitro enzyme assays

LabChip – enzyme kineticsNot covered in this presentation, but also available from PerkinElmer: Radiochemical

Protocol Summary

PreparationOvernight

Histone Extraction

25 min

Detection90 min

Seed cells in white opaqueCulturPlate™-384 microplate

Cell-Histone Lysis buffer15 min

10 min

overnight

Treat cells if desired 4 h (adherent cells)

Acceptor beads (20 µg/mL final) + biotinylated anti-H3 (3 nM final)

60 min

Donor beads (20 µg/mL final)

30 min

Read plate on EnVision® or EnSpire®

Cell-Histone Extraction buffer

Additionvolume

Assayvolume

10 µL 10 µL

5 µL 15 µL

5 µL 20 µL

10 µL 30 µL

10 µL 40 µL

10 µL 50 µL

50 µL

500 untreated cells/well100 NaB-treated cells/well

Cell Titration

0

100

500

1,00

0

2,00

0

5,00

0

10,0

00

0

200,000

400,000

600,000

800,000

1,000,000untreated20mM NaB

Cells per well

Alph

aLIS

A Si

gnal

(cou

nts)

0

100

500

1,00

0

2,00

0

5,00

0

10,0

00

0

200,000

400,000

600,000

800,000

1,000,000

Cells per well

Alph

aLIS

A Si

gnal

(cou

nts)

0

100

500

1,00

0

2,00

0

5,00

0

10,0

00

20,0

00

0

100,000

200,000

300,000

Cells per well

Alph

aLIS

A Si

gnal

(cou

nts)

0

100

500

1,00

0

2,00

0

5,00

0

10,0

00

0

20,000

40,000

60,000

80,000

100,000

120,000

Cells per well

Alph

aLIS

A Si

gnal

(cou

nts)

0

100

500

1,00

0

2,00

0

5,00

0

10,0

00

0

20,000

40,000

60,000

80,000

100,000

120,000

Cells per wellAl

phaL

ISA

Sign

al (c

ount

s)

HeLa HEK 293 Jurkat

OCI-LY-19 SU-DHL-6

Different cell lines exhibit different mark levelsdifferent NaB-fold stimulation

Corroboration of Alpha by Western blot data

Selecting the Right Cell Line

Mol

. Wei

ght (

kDa) 50

403020

1510

3.5

60

- + - + - +NaB

HeLa HEK-293 Jurkat

H3K9ac

Total H3HeLa HEK-293 Jurkat

0

200,000

400,000

600,000

800,000untreated20 mM NaB

Alph

aLIS

A Si

gnal

(cou

nts)

Wigle, SBS 2010 (PKI focus group)

igle, SBS 2010 (PKI focus group)

Wigle, SBS 2010 (PKI focus group)

Wigle, SBS 2010 (PKI focus group)

Wigle, SBS 2010 (PKI focus group)

SummaryAlphaLISA Assays

Significantly less steps, less timeSuperior dynamic range, great sensitivityLow sample volume- 5 l

SureFire Assays- PhosphoProtein DetectionSignificantly less steps, less time vs. WesternsElimination of imaging/ problems with uneven blots

Protein: Protein Interaction AssaysAbility to use large proteins- unlike Fluorescent polarization

or FRETAbility to measure low affinity (mM affinities)

Epigentics AssaysAlpha and TR-FRET assaysMethylation and Acetylation assays

PerkinElmer Contacts

Cathy SweenyReagent Technical Specialiste-mail Catherine.Sweeny@perkinelmer.comPhone: (636) 357-7649

Steven AndersonInstrumentation Sales Specialiste-mail Steven.anderson@perkinelmer.comPhone: (630)414-2827

Maxine SantoroField application Scientiste-mail maxine.santoro@perkinelmer.comPhone: (734) 276-5830

EnSpire AlphaPLUSAlphaLISA/AlphaScreen detection capability

Photometric UV/VIS technology for ELISA assays and DNA/protein quantitation

ELISA reference wavelength correction

Data export in Excel®/text formats to the network or into a USB memory stick

Integrated data‐analysis software: curve fit (lin‐reg, spline, 4PL/5PL), background subtraction, ratio calculation,IC50 calculation, Average, CV %, and Z ‐value

Easy access to the filter wheel with eight barcode identified filter positions

Touch Screen: Easy to use; saves desktop space

Pre‐coded assay protocols

Up to 384‐well plates

21 CFR Part 11 support

Windows® Vista® Operating System