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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 ProteinAntibody SupplierCatalogue No.Membrane UsedSNAP i.d.® 2.0 Dilution Factor or Antibody ConcentrationBlocking Reagent & Antibody DiluentDetection 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 MilliporeSigma. MilliporeSigma 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.