Antibody validation

1289-1
EPITOMICS
sc-1637
sc-7289 sc-937
sc-280
1122-1
SUPPLIERS WEBSITE:
PROTEIN ATLAS WEBSITE:

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Akt1 : n= 2506
NFKB1: n=920 ACTB:n=721
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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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PRKCA : 597
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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)

The specificity problem of antibody array analysis
?

PacOrange
PacBlueAx488
Ax647Ax750
3 3x36=108 3x36x16=1728
AX750 AX488
AX647
AX647
PACBLUE
PAC ORANGE
MAP arrays

Biotin
Streptavidin-
PE
Biotinylated
sample proteins
Color-coded
microsphere
Microsphere Affinity Proteomics
Immobilized antibody

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 21
1,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

150
650
1150
1650
2150
2650
3150
3650
4150
1 3 5 7 9 11
TP53[44](C,O,N)#329ArrSignal2_ 208- 1_ Tween
TP53[44](C,O,N)#329ArrSignal2_ 208- 2_ Tween
TP53[44](C,O,N)#329ArrSignal2_ 208- 3_ TLM
TP53[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_Tween
MAPK1[41](C,N)#421ArrSignal2_ 427- 2_Tween
MAPK1[41](C,N)#421ArrSignal2_ 427- 3_TLM
MAPK1[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_Tween
GAPDH[36](C,M,N)#6417ArrPrg2_ 365- 2_ Tween
GAPDH[36](C,M,N)#6417ArrPrg2_ 365- 3_ TLM
GAPDH[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_ Tween
GRB2[25](C,O,N)#398ArrPrg2_ 193- 2_Tween
GRB2[25](C,O,N)#398ArrPrg2_ 193- 3_TLM
GRB2[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 40ul
PLCG1[149]()#2854ArrPrg2_332- 2_TweenLM 40ul
PLCG1[149]()#2854ArrPrg2_332- 3_Tw 10ul
PLCG1[149]()#2854ArrPrg2_332- 4_Tw LM 10ul
150
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_ TweenLM
40ul
BCAR1[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_TweenLM
40ul
PIK3R1[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_ TweenLM
40ul
AKT1[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

Fraction
12 607 5029 747 350 4086
11 822 4860 858 396 10823
10 688 5831 605 502 10208
9 579 2978 932 1709 682
8 545 1610 5652 3320 500
7 529 1585 10525 1172 384
6 586 3765 624 545 665
5 2193 7604 276 480 664
4 6995 3658 178 473 359
3 9749 556 191 433 294
2 5534 279 196 384 218
1 330 151 182 218 189
Target CDKN2A CDKN1A CDK2 BUB3 RELA
size kDa 17 18 34 37 60
CDKN2A CDKN1A CDK2 BUB3 RELA
Data Table Heat Map

Antibodies sorted
according to target size
20kDa 250kDa
60
70
80
90
110
140
55-60 70 80 90 110
35
40
50
70
60
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_HeLa
2_A431
3_M CF7
4_PBM C
1 3 5 7 9 11
TP53[44](CMitErN)_NBP1_47437_IgG1xG1_ArrNov1_221-
1_HeLa
2_A431
3_MCF7
4_PBMC
s
AB5
1 3 5 7 9 11
TP53[44](CM itErN)_NBX_5134_IgG2a
xG2a_A rrNov1_473-1_HeLa
xG2a_A rrNov1_473-2_A431
xG2a_A rrNov1_473-3_M CF7
xG2a_A rrNov1_473-4_PBM C
AB4
1 3 5 7 9 11
TP53[44](CMitErN)_NBX_5124_IgG1, xG1_ArrNov1_382-
1_HeLa
2_A431
3_M CF7
4_PBMC
1 3 5 7 9 11
TP53[44](CM itErN)_NBX_5132_IgG2b,
xG2b_ArrNov1_501-1_HeLa
xG2b_ArrNov1_501-2_A431
xG2b_ArrNov1_501-3_M CF7
xG2b_ArrNov1_501-4_PBM C
1 3 5 7 9 11
TP53[44](CM itErN)_NBP1_42253_IgG2axG2a_ArrNov1_
457-1_HeLa
457-2_A431
457-3_M CF7
457-4_PBMC
AB1
HeLa
A431
MCF7
PBMC
10% GEL
AB3AB2
1 3 5 7 9 11
1_HeLa
2_A431
3_M CF7
4_PBM C
1 3 5 7 9 11
TP53[44](CM itErN)_NBP1_42165_IgG1xG1_ArrNov1_366-
1_HeLa
2_A431
3_M CF7
4_PBMC
1 3 5 7 9 11
TP53[44](CM itErN)_NBP1_19251_IgGprtAG_ArrNov1_37-
1_HeLa
2_A431
3_M CF7
4_PB MC
SIGNAL
FRACTION
AB6 AB7
AB8 AB9

MS reference data
Fractions
1 12 1 12

R-PE
Test ab
MAP protein arrays:
Target: half of the proteome covered by end 2015
Antibody binding
Beadcolorcode#1
Intended target
Extra reactivity
cDNA from
Joshua LaBaer
University of Arizona
Cell-free expression
GST-tagged proteins
n=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_352-1DIG
CDK2[34]#342Ar106_352-2TWEEN
CDK2[34]#342Ar106_352-3Nucl 450
CDK2[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- 1DIG
CDK2[34]#2634Ar105_150- 2TWEEN
CDK2[34]#2634Ar105_150- 3Nucl 450
CDK2[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
CDK2[34]#2634Ar105_150- 1DIG
CDK2[34]#2634Ar105_150- 2TWEEN
CDK2[34]#2634Ar105_150- 3Nucl 450
CDK2[34]#2634Ar105_150- 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]#342Ar106_352- 1DIG
CDK2[34]#342Ar106_352- 2TWEEN
CDK2[34]#342Ar106_352- 3Nucl 450
CDK2[34]#342Ar106_352- 4LM

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_1dig
NFKB1#412Ar90_261_2tween
NFKB1#412Ar90_261_3nucl
NFKB1#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_1dig
NFKB1#254Ar90_148_2tween
NFKB1#254Ar90_148_3nucl
NFKB1#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- 1dig
CDK4[34]#298Arr132_15- 2tween
CDK4[34]#298Arr132_15- 3NaCl
CDK4[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
CDK4[34]#343Arr132_163- 1dig
CDK4[34]#343Arr132_163- 2tween
CDK4[34]#343Arr132_163- 3NaCl
CDK4[34]#343Arr132_163- 4LM
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
CDK4[34]#205Arr132_35- 1dig
CDK4[34]#205Arr132_35- 2tween
CDK4[34]#205Arr132_35- 3NaCl
CDK4[34]#205Arr132_35- 4LM
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- 1Dig
CCNA2[49]#2696Ar62_94- 2Tween
CCNA2[49]#2696Ar62_94- 3Nucl 140
CCNA2[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- 1Dig
CCNA2[49]#365Arr141_107- 2Tween
CCNA2[49]#365Arr141_107- 3Nucl 140
CCNA2[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
Beadcolorcode#1
Intended target
Extra reactivity

10
100
1000
10000
100000
1 2 3
TP53 CALCA CEACAM8
APOE HES1 YY1
HDLBP ID2 OPRK1
10
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 A431
1ug/ml
1h 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

Antibody validation

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