Antibody validation

1289-1

EPITOMICS

sc-1637sc-7289 sc-937

sc-2801122-1

SUPPLIERS WEBSITE:

PROTEIN ATLAS WEBSITE:

WHICH ANTIBODY WOULD YOU BUY?

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Akt1 : n= 2506 NFKB1: n=920 ACTB:n=721

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Millipore Millipore Millipore Abcam Santa

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Stat1 : n=1027 Stat5A :n=865

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Novus

RAF1: n=1614

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CST1 Sigma1 Abcam Abcam

CDK4 : 1159

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MAPK1 : 1173

The antibody market is dominated by a few products with many citations

#1 #1 #1

#1

#1#1#1

#1 #1

Antibody Impact Factor

Sales

Ab used in publication

More publications

Survival of the first

ANTIBODY CITATION SHARE BY SUPPLIER (2011 DATA)

Survival of the FIRST

Survival of the FINEST

WHY NOT USE ARRAYS?

The specificity problem of antibody array analysis

?

Cheap Chips

The solution

PacOrangePacBlueAx488

Ax647Ax750

3 3x36=108 3x36x16=1728

AX750 AX488

AX

647

AX

647

PA

C B

LUE

PAC ORANGE

MAP arrays

BiotinStreptavidin-

PE

Biotinylated sample proteins

Color-coded microsphere

Microsphere Affinity Proteomics

Immobilized antibody

Biotin-NHS

Native-MAP

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

1 5 9 13 17 21

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

1 5 9 13 17 211,E+01

1,E+02

1,E+03

1,E+04

1,E+05

1 5 9 13 17 21

MS REFERENCE (currently >7000 proteins)

CDK2 Ab1 CHEK1 Ab1

Results obtained by MS

CHEK1 Ab2

Fractions

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1 5 9 13 17 21

CDK2 Ab1

Western MAP

17

150

650

1150

1650

2150

2650

3150

3650

4150

1 3 5 7 9 11

TP53[44](C,O,N)#329ArrSignal2_208- 1_TweenTP53[44](C,O,N)#329ArrSignal2_208- 2_TweenTP53[44](C,O,N)#329ArrSignal2_208- 3_TLMTP53[44](C,O,N)#329ArrSignal2_208- 4_TLM

150

650

1150

1650

2150

2650

3150

3650

4150

1 3 5 7 9 11

MAPK1[41](C,N)#421ArrSignal2_427-1_TweenMAPK1[41](C,N)#421ArrSignal2_427-2_TweenMAPK1[41](C,N)#421ArrSignal2_427-3_TLMMAPK1[41](C,N)#421ArrSignal2_427-4_TLM

150

1150

2150

3150

4150

5150

6150

7150

1 3 5 7 9 11

GAPDH[36](C,M,N)#6417ArrPrg2_365- 1_TweenGAPDH[36](C,M,N)#6417ArrPrg2_365- 2_TweenGAPDH[36](C,M,N)#6417ArrPrg2_365- 3_TLMGAPDH[36](C,M,N)#6417ArrPrg2_365- 4_TLM

150

5150

10150

15150

20150

25150

30150

1 3 5 7 9 11

GRB2[25](C,O,N)#398ArrPrg2_193- 1_TweenGRB2[25](C,O,N)#398ArrPrg2_193- 2_TweenGRB2[25](C,O,N)#398ArrPrg2_193- 3_TLMGRB2[25](C,O,N)#398ArrPrg2_193- 4_TLM

Grb2 21kDa

GAPDH 36kDa

MAPK1 44 kDa

TP53 46kDa

8% GEL

150

1150

2150

3150

4150

5150

6150

1 3 5 7 9 11

PLCG1[149]()#2854ArrPrg2_332- 1_Tween 40ulPLCG1[149]()#2854ArrPrg2_332- 2_TweenLM 40ulPLCG1[149]()#2854ArrPrg2_332- 3_Tw 10ulPLCG1[149]()#2854ArrPrg2_332- 4_Tw LM 10ul

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5150

10150

15150

20150

25150

30150

35150

1 3 5 7 9 11

BCAR1[93](C)#2676ArrSignal2_493- 1_Tween 40ul

BCAR1[93](C)#2676ArrSignal2_493- 2_TweenLM40ulBCAR1[93](C)#2676ArrSignal2_493- 3_Tw 10ul

BCAR1[93](C)#2676ArrSignal2_493- 4_Tw LM 10ul

150

650

1150

1650

2150

2650

3150

3650

4150

4650

1 3 5 7 9 11

PIK3R1[84]()#3721ArrSignal2_408- 1_Tween 40ul

PIK3R1[84]()#3721ArrSignal2_408- 2_TweenLM40ulPIK3R1[84]()#3721ArrSignal2_408- 3_Tw 10ul

PIK3R1[84]()#3721ArrSignal2_408- 4_Tw LM 10ul

150

1150

2150

3150

4150

5150

6150

7150

8150

9150

1 3 5 7 9 11

AKT1[56](C,M,N)#2650ArrPrg2_220- 1_Tween 40ul

AKT1[56](C,M,N)#2650ArrPrg2_220- 2_TweenLM40ulAKT1[56](C,M,N)#2650ArrPrg2_220- 3_Tw 10ul

AKT1[56](C,M,N)#2650ArrPrg2_220- 4_Tw LM 10ul

PLCg 140kDa

BCAR1 110KDa

PI3K 85kDa

Akt1 56 kDa

5% GEL

Fraction12 607 5029 747 350 408611 822 4860 858 396 1082310 688 5831 605 502 10208

9 579 2978 932 1709 6828 545 1610 5652 3320 5007 529 1585 10525 1172 3846 586 3765 624 545 6655 2193 7604 276 480 6644 6995 3658 178 473 3593 9749 556 191 433 2942 5534 279 196 384 2181 330 151 182 218 189

Target CDKN2A CDKN1A CDK2 BUB3 RELAsize kDa 17 18 34 37 60

CDKN2A CDKN1A CDK2 BUB3 RELA

Data Table Heat Map

Antibodies sorted according to target size

20kDa 250kDa

60708090

110

140

55-60 70 80 90 110

35

4050

7060

A western blot with 430 antibodies

8% Gel 5% Gel

1 3 5 7 9 11

TP53[44](CM itErN)_NB200_156_prtAG_ArrNov1_47-1_HeLaTP53[44](CM itErN)_NB200_156_prtAG_ArrNov1_47-2_A431TP53[44](CM itErN)_NB200_156_prtAG_ArrNov1_47-3_M CF7TP53[44](CM itErN)_NB200_156_prtAG_ArrNov1_47-4_PBM C

1 3 5 7 9 11

TP53[44](CM itErN)_NBP1_47437_IgG1xG1_ArrNov1_221-1_HeLaTP53[44](CM itErN)_NBP1_47437_IgG1xG1_ArrNov1_221-2_A431TP53[44](CM itErN)_NBP1_47437_IgG1xG1_ArrNov1_221-3_MCF7TP53[44](CM itErN)_NBP1_47437_IgG1xG1_ArrNov1_221-4_PBMC

s

AB5

1 3 5 7 9 11

TP53[44](CM itErN)_NBX_5134_IgG2axG2a_A rrNov1_473-1_HeLaTP53[44](CM itErN)_NBX_5134_IgG2axG2a_A rrNov1_473-2_A431TP53[44](CM itErN)_NBX_5134_IgG2axG2a_A rrNov1_473-3_M CF7TP53[44](CM itErN)_NBX_5134_IgG2axG2a_A rrNov1_473-4_PBM C

AB4

1 3 5 7 9 11

TP53[44](CM itErN)_NBX_5124_IgG1, xG1_ArrNov1_382-1_HeLaTP53[44](CM itErN)_NBX_5124_IgG1, xG1_ArrNov1_382-2_A431TP53[44](CM itErN)_NBX_5124_IgG1, xG1_ArrNov1_382-3_M CF7TP53[44](CM itErN)_NBX_5124_IgG1, xG1_ArrNov1_382-4_PBMC

1 3 5 7 9 11

TP53[44](CM itErN)_NBX_5132_IgG2b,xG2b_ArrNov1_501-1_HeLaTP53[44](CM itErN)_NBX_5132_IgG2b,xG2b_ArrNov1_501-2_A431TP53[44](CM itErN)_NBX_5132_IgG2b,xG2b_ArrNov1_501-3_M CF7TP53[44](CM itErN)_NBX_5132_IgG2b,xG2b_ArrNov1_501-4_PBM C

1 3 5 7 9 11

TP53[44](CM itErN)_NBP1_42253_IgG2axG2a_ArrNov1_457-1_HeLaTP53[44](CM itErN)_NBP1_42253_IgG2axG2a_ArrNov1_457-2_A431TP53[44](CM itErN)_NBP1_42253_IgG2axG2a_ArrNov1_457-3_M CF7TP53[44](CM itErN)_NBP1_42253_IgG2axG2a_ArrNov1_457-4_PBMC

AB1

HeLaA431MCF7PBMC

10% GELAB3AB2

1 3 5 7 9 11

TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-1_HeLaTP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-2_A 431TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-3_M CF7TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-4_P BM C

1 3 5 7 9 11

TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-1_HeLaTP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-2_A 431TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-3_M CF7TP 53[44](CM itErN)_NBP1_42165_IgG1 xG1_ArrNov1_366-4_P BMC

1 3 5 7 9 11

TP53[44](CM itErN)_NBP1_19251_IgGprtAG_ArrNov1_37-1_HeLaTP53[44](CM itErN)_NBP1_19251_IgGprtAG_ArrNov1_37-2_A431TP53[44](CM itErN)_NBP1_19251_IgGprtAG_ArrNov1_37-3_M CF7TP53[44](CM itErN)_NBP1_19251_IgGprtAG_ArrNov1_37-4_PB MC

SIGNAL

FRACTION

AB6 AB7

AB8 AB9

MS reference data

Fractions1 12 1 12

R-PE

Test ab

MAP protein arrays: Target: half of the proteome covered by end 2015

Antibody binding

Bea

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olor

cod

e #1

Intended target

Extra reactivity

cDNA from Joshua LaBaerUniversity of ArizonaCell-free expressionGST-tagged proteinsn=10K mid 2015

1,E+02

2,E+02

3,E+02

4,E+02

5,E+02

6,E+02

7,E+02

8,E+02

9,E+02

1,E+03

1 5 9 13 17 21

CDK2[34]#342Ar106_352-1DIGCDK2[34]#342Ar106_352-2TWEENCDK2[34]#342Ar106_352-3Nucl 450CDK2[34]#342Ar106_352-4LM

1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21

CDK2[34]#2634Ar105_150- 1DIGCDK2[34]#2634Ar105_150- 2TWEENCDK2[34]#2634Ar105_150- 3Nucl 450CDK2[34]#2634Ar105_150- 4LM

1 Citation680 Citations

1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21


1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21


A highly cited antibody is not necessarily the best reagent

1,E+02

5,E+03

1,E+04

2,E+04

2,E+04

3,E+04

1 5 9 13 17 21

NFKB1#412Ar90_261_1digNFKB1#412Ar90_261_2tweenNFKB1#412Ar90_261_3nuclNFKB1#412Ar90_261_4lm

1,E+02

5,E+03

1,E+04

2,E+04

2,E+04

3,E+04

1 5 9 13 17 21

NFKB1#254Ar90_148_1digNFKB1#254Ar90_148_2tweenNFKB1#254Ar90_148_3nuclNFKB1#254Ar90_148_4lm

1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21

CDK4[34]#298Arr132_15- 1digCDK4[34]#298Arr132_15- 2tweenCDK4[34]#298Arr132_15- 3NaClCDK4[34]#298Arr132_15- 4LM

5

1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21


t

1,E+02

1,E+03

2,E+03

3,E+03

4,E+03

5,E+03

6,E+03

7,E+03

8,E+03

9,E+03

1 5 9 13 17 21


500 Citations 146 Citations

CDK4

1,E+02

6,E+02

1,E+03

2,E+03

2,E+03

3,E+03

1 5 9 13 17 21

CCNA2[49]#2696Ar62_94- 1DigCCNA2[49]#2696Ar62_94- 2TweenCCNA2[49]#2696Ar62_94- 3Nucl 140CCNA2[49]#2696Ar62_94- 4LM

1,E+02

6,E+02

1,E+03

2,E+03

2,E+03

3,E+03

1 5 9 13 17 21

CCNA2[49]#365Arr141_107- 1DigCCNA2[49]#365Arr141_107- 2TweenCCNA2[49]#365Arr141_107- 3Nucl 140CCNA2[49]#365Arr141_107- 4LM

396 0

217 18

Cyclin A

NFKB1

?

?

?

?

What are the extra bands?

R-PE

Test ab

MAP protein arrays: Target: half of the proteome covered by end 2015

Antibody binding

Bea

d c

olor

cod

e #1

Intended target

Extra reactivity

10

100

1000

10000

100000

1 2 3

TP53 CALCA CEACAM8

APOE HES1 YY1

HDLBP ID2 OPRK110

100

1000

10000

100000

1 2 3

TP53 CDK5R1 CALCA

MAPK8 BMP2 HSD3B1

IKZF3 HES1 OPRK1

Testing TP53 mAbs binding at 10 fold dilutions

WB A4311ug/ml1h labeling

Cost-effective antibody validation

Market-leader

Antibody arrays Protein arrays

WB, IHC, IF, ChIP etc

Total cost:10$

50-200$ per assay

High performance antibodies

Market-leader

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Antibody validation