Antibody Optimization with SNAP i.d.® 2.0 System

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Antibody optimization, in the context of Western blotting, is defined as the process of determining which dilution factor of your primary and secondary antibody yields the best signal-to-noise ratio. If you are performing a semiquantitative Western blot, this signal should be in the linear range, neither too faint nor oversaturated.

The SNAP i.d.® 2.0 system can speed up your Western blot process from >4 hours to ~30 minutes. As in many other techniques, each antibody needs to be optimized for the SNAP i.d.® 2.0 system to ensure optimal performance.

How to Adapt Your Western Blotting Protocol for SNAP i.d.® 2.0 Detection Using Antibody Optimization

To save even more of your time, we have created this antibody optimization reference guide with optimized working concentrations for the SNAP i.d.® 2.0 system. This guide will be updated periodically with new working concentrations for a growing list of antibodies.

Chemiluminescent-Based Detection

Target Protein	Antibody Supplier	Catalogue No.	Membrane Used	SNAP i.d.® 2.0 Dilution Factor or Antibody Concentration	Blocking Reagent & Antibody Diluent	Detection Reagent
A12	Abcam	unlisted	PVDF	1:500	Blocker BSA	SuperSignal West Dura
Actin, Beta	Sigma	A5441	Nitrocellulose	1:20000	0.5% milk	ECL Western Blotting Detection Reagent
Actin, Clone AC-15	Sigma	A3854	Nitrocellulose	1:25000	5% milk	SuperSignal West Pico
Actin, Clone C4	Millipore	MAB1501	PVDF	1:2000	Bløk-CH	Immobilon Western HRP Substrate
Actin, Clone C4	Millipore	MAB1501R	Nitrocellulose	1:1000	3% FBS in TBST	SuperSignal West Pico
AKT, phosphoserine (Ser473)	Cell Signaling	4058	Nitrocellulose	1:1000	0.5% milk	ECL Western Blotting Detection Reagent
Akt, phosphotyrosine (Tyr450)	Millipore	07-1643	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
ATR	Millipore	09-070	PVDF	1:500	Bløk-CH	Immobilon Western HRP Substrate
BRCA-1 clone BC70	Millipore	05-842	PVDF	1:500	Bløk-CH	Immobilon Western HRP Substrate
CAF1 p60, clone SS-53, 1-124	Millipore	04-1523	PVDF	1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
Catenin, beta	Millipore	06-734	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
c-Jun, clone 6E4.4	Millipore	05-1076	PVDF	5 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
CREB (Bzip transcription factor)	Millipore	06-863	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
CTTN, Clone 4F11	Millipore	05-180	Nitrocellulose	1:700	0.5% milk	SuperSignal West Dura
CyCA(Cyclophilin A)	Millipore	07-313	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
CYCS	Santa Cruz	SC 7159	Nitrocellulose	1:500	0.5% milk	Pierce ECL Western Blotting Substrate
DLC1	BD Transduction Laboratories	612021	Nitrocellulose	1:1250	5% milk	SuperSignal West Femto
EGFR	Cell Signaling	D38B1	PVDF	1:500	0.5% milk	ECL Western Blotting Detection Reagent
EGFR	Millipore	06-847	PVDF	1:333	0.5% milk	SuperSignal West Dura
Endophilin B1	Millipore	AB10555	PVDF	1:500-1:1000	0.05% Milk	Immobilon Western HRP Substrate
FUBP3	Millipore	07-742	PVDF	2 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
G9a (BAT8)	Millipore	09-071	PVDF	1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
GAPDH	Millipore	MAB374	PVDF	1:8000	Bløk-CH	Immobilon Western HRP Substrate
GAPDH	Santa Cruz	SC25778	PVDF	1:5000	0.5% Milk	Immobilon Western HRP Substrate
GFAP	Millipore	MAB360	PVDF	1:5000	0.5% Milk	ECL Western Blotting Detection Reagent
HCV NS3	Viroge	217-A	Nitrocellulose	1:1000	SuperBlock	Cell Signaling Lumiglo
HCV NS-5a	Biodesign International	C65388M	Nitrocellulose	1:2500	SuperBlock	Cell Signaling Lumiglo
HDAC11	Millipore	09-827	PVDF	0.5 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
Hexim 1	Millipore	07-955	PVDF	4 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
HIF-1 Alpha	BD Biosciences	610598	PVDF	1:1000	0.5% milk	Immuno-Star Chemiluminescence HRP
HLX1	Millipore	09-084	PVDF	2 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
IGF2 mRNA-binding protein 2	Millipore	07-103	PVDF	4 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
IGF2 mRNA-binding protein 3	Millipore	07-104	PVDF	2 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
IKKα	Millipore	07-1007	PVDF	1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
IKKβ	Millipore	07-1008	PVDF	2-10 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
IRS1, clone 58-10C-31	Millipore	05-784R	PVDF	0.1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
JunD	Millipore	07-1334	PVDF	1:2000	Bløk-CH	Immobilon Western HRP Substrate
Lactamase, beta	Millipore	AB3738	PVDF	1:3000	0.5% Milk	Biorad Immune-star Western C Kit
LSM14A	Millipore	ABE37	PVDF	1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
MAP Kinase 1/2 (Erk-1/2)	Millipore	06-182	PVDF	1:500	Bløk-CH	Immobilon Western HRP Substrate
MYPT1, phosphothreonine (Thr696)	Millipore	ABS45	PVDF	0.1 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
Nanog	Millipore	AB5731	PVDF	1:2000	0.5% Milk	Immobilon Western HRP Substrate
NES, Nestin	Millipore	AB5922	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
NeuN, Clone A60	Millipore	MAB377	PVDF	1:1000	5% Milk	Supersignal West Pico
Neurofilament M, C-terminus	Millipore	AB1987	PVDF	1:2000	5% Milk	ECL Plus Western Blotting Detection Reagent
NFEL, 70KDa cloneDA2	Millipore	MAB1615	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
NFkB p50	Millipore	06-886	Nitrocellulose	1:1000	SuperBlock	Cell Signaling Lumiglo
NMDAR 3A	Millipore	07-356	PVDF	1:5000	3% milk	Immun-Star HRP Substrate
Nrf2	Santa Cruz	SC 13032	PVDF	1:750	3% milk	ECL Western Blotting Detection Reagent
Oct4	Millipore	AB3209	PVDF	1:4000	0.5% milk	Immobilon Western HRP Substrate
P38/SAP K2	Millipore	05-454	PVDF	1:600	Bløk-CH	Immobilon Western HRP Substrate
P-53	Millipore	AB565	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
PAM	Santa Cruz	SC-17393	PVDF	1:5000	0.5% Milk	Supersignal West Pico
PLCγ-1, phosphotyrosine (Tyr783)	Millipore	07-2134	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
PLK1, phosphoserine (Ser137)	Millipore	07-1348	PVDF	0.5 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
PRMT5	Millipore	07-405	Nitrocellulose	1:1000	1% milk	Supersignal West Pico
PSD95	Thermo Fisher Scientific	MA1-046	PVDF	1:1000	3% BSA	Immobilon Western HRP Substrate
RAC1	BD Transduction Laboratories	610650	Nitrocellulose	1:1250	0.5% milk	SuperSignal West Dura
Rac1, Clone 23A8	Millipore	05-389	PVDF	1:5000	0.5% milk	Immobilon Western HRP Substrate
RelA	Cell Signaling	3033s	PVDF	1:3000	0.5% Milk	ECL Western Blotting Detection Reagent
RNF110/PCGF2	Millipore	09-724	PVDF	2 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
RNF2 (Ring1b)	Millipore	09-723	PVDF	1.5 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
RPA2 p34, clone RPA20 1-46	Millipore	04-1481	PVDF	0.5 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
RSKA-1/S6 Kinase	Cell Signaling	9205	PVDF	1:2000	PO4 Western Block (.1% gelatin, 3% BSA in 1XTBST)	ECL Plus Western Blotting Detection Reagent
Sox2	Millipore	AB5603	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
Sox2	Millipore	AB5603	PVDF	1:2000	0.5% milk	Immobilon Western HRP Substrate
SP1	Millipore	07-645	Nitrocellulose	10μg	5% milk	ECL Western Blotting Detection Reagent
STAT 1	Millipore	05-987	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
STAT 3	Cell Signaling	9145	Nitrocellulose	1:300	0.5% BSA	ECL Plus Western Blotting Detection Reagent
STAT 3	Millipore	06-596	Nitrocellulose	1:300	SuperBlock	Cell Signaling Lumiglo
SYP	Millipore	MAB368	PVDF	1:1000	Bløk-CH	Immobilon Western HRP Substrate
TCR B-Chain	Santa Cruz	SC 9101	Nitrocellulose	1:1000	0.5% Milk	Pierce ECL Western Blotting Substrate
TIMP-1, C-terminus	Millipore	AB6007	PVDF	1:500	Bløk-CH	Immobilon Western HRP Substrate
Tubulin beta III	Millipore	CBL412	PVDF	1:200	0.5% Milk	Immobilon Western HRP Substrate
UBX domain-containing protein 1 (UBXN1/SAKS1)	Millipore	AB10041	PVDF	2 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
Unc84B/SUN2	Millipore	06-1038	PVDF	1:250	Bløk-CH	Immobilon Western HRP Substrate
VIM	Millipore	AB1620	PVDF	1:200	Bløk-CH	Immobilon Western HRP Substrate
VIPR1	Genscript	n/a	Nitrocellulose	3 μg/ml	0.4% milk	ECL Plus Western Blotting Detection Reagent
XBP1	Millipore	09-722	PVDF	6 μg/ml	0.05% Milk	Immobilon Western HRP Substrate
Target PMT*
Nitrotyrosine	Millipore	06-284	PVDF	1:2000	0.5% milk	Immobilon Western HRP Substrate
Phosphotyrosine	Millipore	4G10	PVDF	1:4000	Bløk-PO	Immobilon Western HRP Substrate
Target Tag
Flag Tag	Sigma Aldrich	A8592	PVDF	1:500	0.5% milk	Supersignal West Pico Luminescent Reagent
HA (Hemagglutinin epitope tag, YPYDVPDYA)	Covance	MMS-101P	Nitrocellulose	1:1000	0.5% milk	ECL Western Blotting Detection Reagents
His Tag, Clone 4D11	Millipore	05-531	PVDF	1:1000	0.5% milk	Immobilon Western HRP Substrate

Notes:
¹ All milk blocker were dissolved in TBS-T
² We do not recommend using milk for phosphorylation-based detection as milk contains phosphorylated proteins
³ Recommended concentration of NFDM: ≤ 0.5%
* Post Translational Modification

Some of the optimal incubation condition information for the antibodies listed above was collected from public sources and has not been validated by Merck. Merck offers no warranty, express or implied, regarding the accuracy or completeness of this information and accepts no liability for unsatisfactory results. Each end-user should conduct its own analysis to determine an appropriate course of action with respect to this information.