Western Blotting Tools
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Millipore, Advancing Life Science Together, SNAP i.d., Immobilon, Amicon, and Ultracel are registered trademarks of Millipore Corporation. ReNcell, PureProteome, Luminata, Bløk, ReBlot, ChemiLucent, and the M mark are trademarks of Millipore Corporation. Qdot is a trademark of Quantum Dot Corporation. Kodak is a registered trademark of Eastman Kodak Company. Lit. No. PB1033EN00 Printed in the USA 03/10 LS-SBU-10-02831 ©2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.
Immobilon Transfer MembranesDescription Size Qty/Pk Catalogue No.
Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850
26.5 cm x 3.75 m 1 roll IPVH00010
Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850
26.5 cm x 3.75 m 1 roll ISEQ00010
Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810
26.5 cm x 3.75 m 1 roll IPFL00010
SNAP i.d. System
Description Components Qty/Pk Catalogue No.
SNAP i.d. Protein Detection System WBAVDBASE
SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01
Double Blot Holder 30/pk WBAVDBH02
Triple Blot Holder 20/pk WBAVDBH03
Antibody Collection Tray 20/pk WBAVDABTR
SNAP i.d. Blot Roller 1/pk WBAVDR0LL
Bløk Noise-Canceling ReagentsDescription Detection Method Qty/Pk Catalogue No.
Bløk-CH Reagent Chemiluminescence Detection 500 mL/bottle WBAVDCH01
Bløk-FL Reagent Fluorescence Detection 500 mL/bottle WBAVDFL01
Bløk-PO Reagent Phosphoprotein Detection 500 mL/bottle WBAVDP001
Luminata Western HRP SubstratesDescription Qty/Pk Catalogue No.
Luminata Classico Western HRP Substrate
500 mL WBLUC0500
Luminata Crescendo Western HRP Substrate
500 mL WBLUR0500
Luminata Forte Western HRP Substrate
500 mL WBLUF0500
Western Blotting Enhancing ReagentsDescription Qty/Pk Catalogue No.
ChemiLucent Plus Western Blot Enhancing Kit
1 kit 2650
ReBlot Plus Mild Antibody Stripping Solution, 10x
50 mL 2502
ReBlot Plus Strong Antibody Stripping Solution, 10x
50 mL 2504
ORdERINg INfORMATION
Western Blotting ToolsPublication Quality Westerns in Minuteswith Millipore’s Pre-optimized Products
TO PLACE AN ORdER In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)
In Europe, please call Customer Service:
France: 0825.045.645 • Spain: 901.516.645 Option 1 • Germany: 01805.045.645 • Italy: 848.845.645 • UK: 0870.900.46.45
For other countries across Europe and the world, please visit www.millipore.com/offices.
For Technical Service, please visit www.millipore.com/techservice.
www.millipore.com/WBtools
3 3
One of the greatest challenges in advancing research is obtaining
consistent, quality results. In Western blotting, the most important
factor in determining the success of experiments is the quality of
resources used, including the protein extraction kit, transfer membrane
and reagents. Millipore offers an array of Western blotting products
that are pre-optimized to work synergistically, providing strong specific
signals and low background to help you quickly produce publication
quality results.
Ready, set, publish! At each step in the Western blotting workflow,
choose the Millipore product with unique advantages that will drive
your research forward.
Millipore’s Western blotting portfolio delivers the highest quality in the shortest time, setting a new pace for discovery.
ZERO TO PuBLICATION QuALITy WESTERNS IN Protein Extraction &
Sample Preparation Protein extraction and purification represent the first of many challenges in obtaining intact, active proteins.
Millipore’s quality reagents unite superior performance with speed to reduce exposure of proteins to unfavorable
conditions, leading to more stable, intact proteins for downstream analysis.
Extraction Kits and Protease InhibitorsProtein stability is fundamental to all aspects of protein research, including analysis by Western blotting. Combine our gentle protein extraction kits with protease inhibitors to obtain stabilized, intact, and active proteins.
Description Qty/Pk Catalogue No.
Compartment Protein Extraction Kit 1 kit 2145
Total Protein Extraction Kit 1 kit 2140
Nuclear Extraction Kit 100 assays 2900
RIPA Lysis Buffer, 10X 100 mL 20-188
Protease Inhibitor Cocktail 1 vial 20-201
Chymostatin 100 mg EI6
Leupeptin 100 mg EI8
Pepstatin A 100 mg EI10
Affinity PurificationPurify your protein with PureProteome magnetic beads or agarose beads. PureProteome magnetic beads ensure fast, effective isolation of proteins without sample loss and are available in nickel, protein A, or protein G formats.
Description Qty/Pk Catalogue No.
PureProteome Nickel Magnetic Beads 10 mL LSKMAGH10
PureProteome Protein A Magnetic Beads 10 mL LSKMAGA10
PureProteome Protein G Magnetic Beads 10 mL LSKMAGG10
Protein A Agarose, fast flow 10 mL 16-156
Protein G Agarose, fast flow 10 mL 16-266
Streptavidin Agarose Conjugate 10 mL 16-126
Buffer Exchange and ConcentrationSimultaneously concentrate and desalt your samples with Amicon Ultra centrifugal filters. Their unparalleled rapid and reproducible performance minimizes protein exposure to harsh buffers.
Description Qty/Pk Catalogue No.
Amicon Ultra - 0.5 mL Filters* 24/pk UFC501024
96/pk UFC501096
Amicon Ultra - 4 mL Filters* 24/pk UFC801024
96/pk UFC801096
Amicon Ultra - 15 mL Filters* 24/pk UFC901024
96/pk UFC901096
*10,000 MWCO. For additional MWCOs, visit www.millipore.com or contact Technical Service.
Protein Extraction & Preparation Blocking detection
Electrophoresis & Transfer
Antibody Incubation,
Washing
Gentle protein extraction kits, pg. 3
Rapid protein isolation with PureProteome™ magnetic beads, pg. 3
Fast, effective concentration with Amicon® Ultra centrifugal filters, pg. 3
High protein binding with Immobilon® membranes, pg. 4
22-minute immunodetection with SNAP i.d.® protein detection system, pg. 6
Protein-free Bløk™ noise-cancelling reagents, pg. 8
SNAP i.d. protein detection system,pg. 6
Optimized antibodies for the SNAP i.d. detection system, pg. 9
Premixed Luminata™ Western HRP substrates for stronger signals, pg. 10
Protein Extraction & Preparation
22MINuTES!
3 3
One of the greatest challenges in advancing research is obtaining
consistent, quality results. In Western blotting, the most important
factor in determining the success of experiments is the quality of
resources used, including the protein extraction kit, transfer membrane
and reagents. Millipore offers an array of Western blotting products
that are pre-optimized to work synergistically, providing strong specific
signals and low background to help you quickly produce publication
quality results.
Ready, set, publish! At each step in the Western blotting workflow,
choose the Millipore product with unique advantages that will drive
your research forward.
Millipore’s Western blotting portfolio delivers the highest quality in the shortest time, setting a new pace for discovery.
ZERO TO PuBLICATION QuALITy WESTERNS IN Protein Extraction &
Sample Preparation Protein extraction and purification represent the first of many challenges in obtaining intact, active proteins.
Millipore’s quality reagents unite superior performance with speed to reduce exposure of proteins to unfavorable
conditions, leading to more stable, intact proteins for downstream analysis.
Extraction Kits and Protease InhibitorsProtein stability is fundamental to all aspects of protein research, including analysis by Western blotting. Combine our gentle protein extraction kits with protease inhibitors to obtain stabilized, intact, and active proteins.
Description Qty/Pk Catalogue No.
Compartment Protein Extraction Kit 1 kit 2145
Total Protein Extraction Kit 1 kit 2140
Nuclear Extraction Kit 100 assays 2900
RIPA Lysis Buffer, 10X 100 mL 20-188
Protease Inhibitor Cocktail 1 vial 20-201
Chymostatin 100 mg EI6
Leupeptin 100 mg EI8
Pepstatin A 100 mg EI10
Affinity PurificationPurify your protein with PureProteome magnetic beads or agarose beads. PureProteome magnetic beads ensure fast, effective isolation of proteins without sample loss and are available in nickel, protein A, or protein G formats.
Description Qty/Pk Catalogue No.
PureProteome Nickel Magnetic Beads 10 mL LSKMAGH10
PureProteome Protein A Magnetic Beads 10 mL LSKMAGA10
PureProteome Protein G Magnetic Beads 10 mL LSKMAGG10
Protein A Agarose, fast flow 10 mL 16-156
Protein G Agarose, fast flow 10 mL 16-266
Streptavidin Agarose Conjugate 10 mL 16-126
Buffer Exchange and ConcentrationSimultaneously concentrate and desalt your samples with Amicon Ultra centrifugal filters. Their unparalleled rapid and reproducible performance minimizes protein exposure to harsh buffers.
Description Qty/Pk Catalogue No.
Amicon Ultra - 0.5 mL Filters* 24/pk UFC501024
96/pk UFC501096
Amicon Ultra - 4 mL Filters* 24/pk UFC801024
96/pk UFC801096
Amicon Ultra - 15 mL Filters* 24/pk UFC901024
96/pk UFC901096
*10,000 MWCO. For additional MWCOs, visit www.millipore.com or contact Technical Service.
Protein Extraction & Preparation Blocking detection
Electrophoresis & Transfer
Antibody Incubation,
Washing
Gentle protein extraction kits, pg. 3
Rapid protein isolation with PureProteome™ magnetic beads, pg. 3
Fast, effective concentration with Amicon® Ultra centrifugal filters, pg. 3
High protein binding with Immobilon® membranes, pg. 4
22-minute immunodetection with SNAP i.d.® protein detection system, pg. 6
Protein-free Bløk™ noise-cancelling reagents, pg. 8
SNAP i.d. protein detection system,pg. 6
Optimized antibodies for the SNAP i.d. detection system, pg. 9
Premixed Luminata™ Western HRP substrates for stronger signals, pg. 10
Protein Extraction & Preparation
22MINuTES!
4 5
Immobilon Western Blotting Transfer Membranes
The signal intensity in a Western blot is highly dependent on the
protein’s density after a Western transfer. When an
insufficient amount of protein is bound to the
membrane, a strong specific signal can be very
difficult to obtain. For that reason, the quality of
the Western blotting transfer membrane
is essential to obtaining high signal-
to-noise ratios and clear experimental
results.
The PVDF Immobilon membranes provide high
protein binding capacity resulting in strong signals.
Each of the Immobilon membranes has been optimized for a
different protein blotting application.
Immobilon-Ptransfer membrane
Immobilon-PSQ
transfer membraneImmobilon-FLtransfer membrane
description Optimized to bind proteins transferred from a variety of gel matrices
Uniform pore structure results in superior binding of proteins with MW <20 kDa
Optimized for fluorescence immunodetection applications
Composition PVDF PVDF PVDF
Pore size 0.45 µm 0.2 µm 0.45 µm
Phobicity Hydrophobic Hydrophobic Hydrophobic
Applications • Western blotting• Binding assays• Amino acid analysis• N-terminal protein sequencing• Dot/slot blotting• Glycoprotein visualization• Lipopolysaccharide analysis• Mass spectrometry
• Low molecular weight Western blotting• Amino acid analysis• Mass spectrometry• N-terminal protein sequencing
• Western blotting• Dot/slot blotting• Fluorescence immunodetection
detection methods • Chromogenic• Chemiluminescent• Radioactive
• Chromogenic• Chemiluminescent• Radioactive
• Fluorescent• Chromogenic• Chemifluorescent• Chemiluminescent
Protein binding capacity Insulin: 160 µg/cm2
BSA: 215 µg/cm2
Goat IgG: 294 µg/cm2
Insulin: 262 µg/cm2
BSA: 340 µg/cm2
Goat IgG: 448 µg/cm2
Insulin: 155 µg/cm2
BSA: 205 µg/cm2
Goat IgG: 300 µg/cm2
Comparison of Immobilon membrane properties
Multiplex detection using fluorescent probes on the Immobilon FL membrane Actin-tubulin assay on Immobilon-FL membrane. Rabbit muscle actin (red) was detected using rabbit anti-actin secondary antibodies and QDot® 655 goat anti-rabbit secondary antibodies. Porcine brain tubulin (green) was detected using mouse anti-tubulin primary and QDot 565 goat anti-mouse secondary antibodies. Sensitivities down to 1 ng were observed on a Kodak® imager. Data provided by Quantum Dot Corporation.
Description Size Qty/Pk Catalogue No.
Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850
26.5 cm x 3.75 m 1 roll IPVH00010
Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850
26.5 cm x 3.75 m 1 roll ISEQ00010
Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810
26.5 cm x 3.75 m 1 roll IPFL00010
ORdERINg INfORMATION
For a complete listing of available Immobilon membranes, visit www.millipore.com/immobilonwestern.
Immobilon PSQ membrane prevents the proteins from blowing through the membrane, increasing protein signal
Molecular weight standards (lanes 1 and 3) and calf liver lysate (lanes 2 and 4) were transferred to Immobilon-P or Immobilon-PSQ membranes. A sheet of Immobilon-PSQ membrane was placed behind the primary membranes to capture proteins that passed through (lanes 5 and 6 behind Immobilon-P membrane; lanes 7 and 8 behind Immobilon-PSQ membrane).
200
116976655
3631
21.5
14.4
6
3.5
kDa 1 2
P PSQ PSQ Backup
3 4 5 6 7 8
1 ng1000 ng
1000 ng1 ng
Electrophoresis & Transfer
KEy fEATuRES• High protein binding capacity
• Easy to strip and reprobe
• High tensile strength and flexibility
• Highly resistant to organic solvents
Validated for the SNAP i.d. system
4 5
Immobilon Western Blotting Transfer Membranes
The signal intensity in a Western blot is highly dependent on the
protein’s density after a Western transfer. When an
insufficient amount of protein is bound to the
membrane, a strong specific signal can be very
difficult to obtain. For that reason, the quality of
the Western blotting transfer membrane
is essential to obtaining high signal-
to-noise ratios and clear experimental
results.
The PVDF Immobilon membranes provide high
protein binding capacity resulting in strong signals.
Each of the Immobilon membranes has been optimized for a
different protein blotting application.
Immobilon-Ptransfer membrane
Immobilon-PSQ
transfer membraneImmobilon-FLtransfer membrane
description Optimized to bind proteins transferred from a variety of gel matrices
Uniform pore structure results in superior binding of proteins with MW <20 kDa
Optimized for fluorescence immunodetection applications
Composition PVDF PVDF PVDF
Pore size 0.45 µm 0.2 µm 0.45 µm
Phobicity Hydrophobic Hydrophobic Hydrophobic
Applications • Western blotting• Binding assays• Amino acid analysis• N-terminal protein sequencing• Dot/slot blotting• Glycoprotein visualization• Lipopolysaccharide analysis• Mass spectrometry
• Low molecular weight Western blotting• Amino acid analysis• Mass spectrometry• N-terminal protein sequencing
• Western blotting• Dot/slot blotting• Fluorescence immunodetection
detection methods • Chromogenic• Chemiluminescent• Radioactive
• Chromogenic• Chemiluminescent• Radioactive
• Fluorescent• Chromogenic• Chemifluorescent• Chemiluminescent
Protein binding capacity Insulin: 160 µg/cm2
BSA: 215 µg/cm2
Goat IgG: 294 µg/cm2
Insulin: 262 µg/cm2
BSA: 340 µg/cm2
Goat IgG: 448 µg/cm2
Insulin: 155 µg/cm2
BSA: 205 µg/cm2
Goat IgG: 300 µg/cm2
Comparison of Immobilon membrane properties
Multiplex detection using fluorescent probes on the Immobilon FL membrane Actin-tubulin assay on Immobilon-FL membrane. Rabbit muscle actin (red) was detected using rabbit anti-actin secondary antibodies and QDot® 655 goat anti-rabbit secondary antibodies. Porcine brain tubulin (green) was detected using mouse anti-tubulin primary and QDot 565 goat anti-mouse secondary antibodies. Sensitivities down to 1 ng were observed on a Kodak® imager. Data provided by Quantum Dot Corporation.
Description Size Qty/Pk Catalogue No.
Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850
26.5 cm x 3.75 m 1 roll IPVH00010
Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850
26.5 cm x 3.75 m 1 roll ISEQ00010
Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810
26.5 cm x 3.75 m 1 roll IPFL00010
ORdERINg INfORMATION
For a complete listing of available Immobilon membranes, visit www.millipore.com/immobilonwestern.
Immobilon PSQ membrane prevents the proteins from blowing through the membrane, increasing protein signal
Molecular weight standards (lanes 1 and 3) and calf liver lysate (lanes 2 and 4) were transferred to Immobilon-P or Immobilon-PSQ membranes. A sheet of Immobilon-PSQ membrane was placed behind the primary membranes to capture proteins that passed through (lanes 5 and 6 behind Immobilon-P membrane; lanes 7 and 8 behind Immobilon-PSQ membrane).
200
116976655
3631
21.5
14.4
6
3.5
kDa 1 2
P PSQ PSQ Backup
3 4 5 6 7 8
1 ng1000 ng
1000 ng1 ng
Electrophoresis & Transfer
KEy fEATuRES• High protein binding capacity
• Easy to strip and reprobe
• High tensile strength and flexibility
• Highly resistant to organic solvents
Validated for the SNAP i.d. system
6 7
SNAP i.d. Protein detection SystemRapid immunodetection in just 22 minutes!
Western blotting has been the life scientist’s workhorse since its introduction in
1979, and it remains the gold standard in protein detection. Few improvements
were made to this laborious, time-consuming technique, until the SNAP i.d.
protein detection system was introduced in 2008. The SNAP i.d. system
decreases the immunodetection phase of Western blotting to 22 minutes by
using two mechanisms to favor antibody-antigen interaction:
1. The SNAP i.d. protocol uses higher antibody (Ab) concentrations
relative to traditional methods, driving antibody (Ab)-antigen (Ag)
complex formation.
2. The SNAP i.d. system uses vacuum suction to pull the antibodies through the membrane, exposing all of the membrane-
embedded target proteins to the antibody. This increases the available antigen concentration, driving the equilibrium to
favor antibody-antigen complex formation.
KEy fEATuRES• 22-minute immunodetection
enabling more experiments
in less time
• Increased antibody-antigen binding
• Superior washes for lower
background
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
DETECTION OF TRANSFERRINBlots of a serial dilution of human serum were probed with anti-human transferrin followed by anti-sheep rabbit HRP-conjugated IgG secondary antibody (AP147P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF VIMENTINBlots of a serial dilution of ReNcell™ CX cell lysates (14, 7, 3 µg) were probed with anti-human vimentin (AB1620, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP106P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF MAP KINASE 1/2 (ERK 1/2)Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) were probed with anti-MAP Kinase 1/2 (06-182, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP132P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF ADENOVIRUSBlots of a serial dilution of Adenovirus infected HEK cells were probed with anti-Adenovirus clone 20/11 (MAB8052, Millipore) followed by anti-goat mouse HRP-conjugated IgG secondary antibody (AP124P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
Traditional Western Blot
77 kDa
58 kDa
44 kDa
140 kDa
P=1:80,000S=1:50,000
P=1:20,000S=1:80,000
P=1:1,000S=1:200,000
P=1:5,000S=1:10,000
SNAP i.d. System Conditions
P=1:10,000S=1:20,000
P=1:1,000S=1:10,000
P=1:200S=1:40,000
P=1:1,000S=1:10,000
A.
B.
C.
D.
ORdERINg INfORMATIONDescription Components Qty/Pk Catalogue No.
SNAP i.d. Protein Detection System WBAVDBASE
SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01Double Blot Holder 30/pk WBAVDBH02Triple Blot Holder 20/pk WBAVDBH03Antibody Collection Tray 20/pk WBAVDABTRSNAP i.d. Blot Roller 1/pk WBAVDR0LL
BlockingAntibody Incubation, Washing
During electrotransfer of proteins from SDS-PAGE gels to membranes, proteins get trapped within the membrane’s 3-dimensional structure. Traditional Western blotting relies on the diffusion of the antibody through the membrane to reach entrapped proteins. This is a very slow and inefficient process. The SNAP i.d. system actively pulls antibodies through the membrane, increasing their exposure to the entrapped proteins and decreasing the required antibody incubation time.
Comparison of Westerns using the SNAP i.d. system and traditional immunoblotting
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
[Ab] + [Ag] [Ab Ag]
[Ab] + [Ag] [Ab Ag]
•
•
[Ab] + [Ag] [Ab Ag]
[Ab] + [Ag] [Ab Ag]
•
•
6 7
SNAP i.d. Protein detection SystemRapid immunodetection in just 22 minutes!
Western blotting has been the life scientist’s workhorse since its introduction in
1979, and it remains the gold standard in protein detection. Few improvements
were made to this laborious, time-consuming technique, until the SNAP i.d.
protein detection system was introduced in 2008. The SNAP i.d. system
decreases the immunodetection phase of Western blotting to 22 minutes by
using two mechanisms to favor antibody-antigen interaction:
1. The SNAP i.d. protocol uses higher antibody (Ab) concentrations
relative to traditional methods, driving antibody (Ab)-antigen (Ag)
complex formation.
2. The SNAP i.d. system uses vacuum suction to pull the antibodies through the membrane, exposing all of the membrane-
embedded target proteins to the antibody. This increases the available antigen concentration, driving the equilibrium to
favor antibody-antigen complex formation.
KEy fEATuRES• 22-minute immunodetection
enabling more experiments
in less time
• Increased antibody-antigen binding
• Superior washes for lower
background
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
DETECTION OF TRANSFERRINBlots of a serial dilution of human serum were probed with anti-human transferrin followed by anti-sheep rabbit HRP-conjugated IgG secondary antibody (AP147P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF VIMENTINBlots of a serial dilution of ReNcell™ CX cell lysates (14, 7, 3 µg) were probed with anti-human vimentin (AB1620, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP106P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF MAP KINASE 1/2 (ERK 1/2)Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) were probed with anti-MAP Kinase 1/2 (06-182, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP132P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
DETECTION OF ADENOVIRUSBlots of a serial dilution of Adenovirus infected HEK cells were probed with anti-Adenovirus clone 20/11 (MAB8052, Millipore) followed by anti-goat mouse HRP-conjugated IgG secondary antibody (AP124P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).
Traditional Western Blot
77 kDa
58 kDa
44 kDa
140 kDa
P=1:80,000S=1:50,000
P=1:20,000S=1:80,000
P=1:1,000S=1:200,000
P=1:5,000S=1:10,000
SNAP i.d. System Conditions
P=1:10,000S=1:20,000
P=1:1,000S=1:10,000
P=1:200S=1:40,000
P=1:1,000S=1:10,000
A.
B.
C.
D.
ORdERINg INfORMATIONDescription Components Qty/Pk Catalogue No.
SNAP i.d. Protein Detection System WBAVDBASE
SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01Double Blot Holder 30/pk WBAVDBH02Triple Blot Holder 20/pk WBAVDBH03Antibody Collection Tray 20/pk WBAVDABTRSNAP i.d. Blot Roller 1/pk WBAVDR0LL
BlockingAntibody Incubation, Washing
During electrotransfer of proteins from SDS-PAGE gels to membranes, proteins get trapped within the membrane’s 3-dimensional structure. Traditional Western blotting relies on the diffusion of the antibody through the membrane to reach entrapped proteins. This is a very slow and inefficient process. The SNAP i.d. system actively pulls antibodies through the membrane, increasing their exposure to the entrapped proteins and decreasing the required antibody incubation time.
Comparison of Westerns using the SNAP i.d. system and traditional immunoblotting
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
Traditional Western BlotTraditional Western blotting
relies on diffusionThe SNAP i.d. system actively pulls
the reagents through the membrane
SNAP i.d. System
Membrane surface
Entrapped protein
[Ab] + [Ag] [Ab Ag]
[Ab] + [Ag] [Ab Ag]
•
•
[Ab] + [Ag] [Ab Ag]
[Ab] + [Ag] [Ab Ag]
•
•
8 9
Bløk Noise-Cancelling Reagents
Optimized Antibodies for the SNAP i.d. System Obtain fast, reproducible results using antibodies optimized for the SNAP i.d. systemBløk reagents are a family of uniquely formulated protein-free blocking solutions
that provide numerous benefits including:
• Reduced background for chemiluminescent or fluorescent detection.
• Unique formulation for detection of phosphorylated proteins.
• More stable diluent for antibodies than milk.
• Allows chromogenic staining of membranes after immunodetection.
• Engineered for superior reagent flow through SNAP i.d. blot holders.
Target Protein Catalogue No.SNAP i.d. Dilution Factor or Antibody Concentration
Actin, clone C4 MAB1501 1:2000
Akt, phosphotyrosine (Tyr450) 07-1643 1:1000
ATR 09-070 1:500
BRCA-1 clone BC70 05-842 1:500
CAF1 p60, clone SS-53, 1-124 04-1523 1 µg/mL
Catenin, b 06-734 1:200
c-Jun, clone 6E4.4 05-1076 5 µg/mL
CREB (bZIP transcription factor) 06-863 1:200
CyPA (Cyclophilin A) 07-313 1:1000
Endophilin B1 AB10555 1:500-1:1000
FUBP3 07-742 2 µg/mL
G9a (BAT8) 09-071 1 µg/mL
GAPDH MAB374 1:8000
HDAC11 09-827 0.5 µg/mL
Hexim 1 07-955 4 µg/mL
HLX1 09-084 2 µg/mL
IGF2 mRNA-binding protein 2 07-103 4 µg/mL
IGF2 mRNA-binding protein 3 07-104 2 µg/mL
IKKa 07-1007 1 µg/mL
IKKb 07-1008 2-10 µg/mL
IRS1, clone 58-10C-31 05-784R 0.1 µg/mL
JunD 07-1334 1:2000
LSM14A ABE37 1 µg/mL
MAP Kinase 1/2 (Erk-1/2) 06-182 1:500
MYPT1, phosphothreonine (Thr696) ABS45 0.1 µg/mL
NES, Nestin AB5922 1:1000
NEFL, 70 kDa clone DA2 MAB1615 1:200
P38/SAP-K2 05-454 1:600
P 53 AB565 1:1000
PLCg-1, phosphotyrosine (Tyr783) 07-2134 1:1000
PLK1, phosphoserine (Ser137) 07-1348 0.5 µg/mL
Post-immunodetection chromogenic staining of blots When weak signals were obtained in lanes 5 & 6, blots were stained with Coomassie blue following either chemiluminescence (left blots) or fluorescence (right blots) detection to verify equal loading and protein transfer of all lanes.
Lane 1: Molecular weight marker Lane 2: A431 Pervanadate (PVD) stimulated Lane 3: A431 control non stimulated
(fresh samples)Lane 4: A431 EGFR stimulated
(fresh samples)Lane 5: A431 control non stimulated
(old samples)Lane 6: A431 EGFR stimulated
(old samples)
1 2 3 4 5 6 1 2 3 4 5 6
Chemiluminescence detection
Blot stained after detection
1 2 3 4 5 6 1 2 3 4 5 6
Fluorescence detection
Blot stained after detection
ORdERINg INfORMATIONDescription Detection Method Qty/Pk Catalogue No.
Bløk-CH Reagent Chemiluminescence detection 500 mL/bottle WBAVDCH01
Bløk-FL Reagent Fluorescence detection 500 mL/bottle WBAVDFL01
Bløk-PO Reagent Phosphoprotein detection 500 mL/bottle WBAVDP001
BlockingAntibody Incubation
Bløk-PO reagent preserves the phosphorylation state of the protein
Chemiluminescence detection of pERK in EGF-stimulated A431 lysate (10-2.5 µg/lane,12-110, Millipore) using anti-pERK antibody (1:200, 05-797R, Millipore). Bands were detected using Luminata Forte Western HRP substrate (WBLUF0500, Millipore). pERK phosphorylated doublet indicated by arrow. NFDM - Non-fat dry milk
Competitor L Competitor P NFDM Bløk – PO
SNAP i.d. PublicationsMihrshahi R, Barclay AN, Brown MH. J Immunol. 2009 Oct 15; 183(8):4879-86.
Fujimori K, Ueno T, et al. J Biol Chem. 2010 Mar 19; 285(12):8880-6.
Sakane A, Honda K, Sasaki T. Mol Cell Biol. 2010 Feb; 30(4):1077-87.
Many more to be found on www.millipore.com/SnapPub.
Validated for the SNAP i.d. system
For a complete listing, visit the SNAP i.d. Antibody Optimization Reference Guide at www.millipore.com/SNAPab.
8 9
Bløk Noise-Cancelling Reagents
Optimized Antibodies for the SNAP i.d. System Obtain fast, reproducible results using antibodies optimized for the SNAP i.d. systemBløk reagents are a family of uniquely formulated protein-free blocking solutions
that provide numerous benefits including:
• Reduced background for chemiluminescent or fluorescent detection.
• Unique formulation for detection of phosphorylated proteins.
• More stable diluent for antibodies than milk.
• Allows chromogenic staining of membranes after immunodetection.
• Engineered for superior reagent flow through SNAP i.d. blot holders.
Target Protein Catalogue No.SNAP i.d. Dilution Factor or Antibody Concentration
Actin, clone C4 MAB1501 1:2000
Akt, phosphotyrosine (Tyr450) 07-1643 1:1000
ATR 09-070 1:500
BRCA-1 clone BC70 05-842 1:500
CAF1 p60, clone SS-53, 1-124 04-1523 1 µg/mL
Catenin, b 06-734 1:200
c-Jun, clone 6E4.4 05-1076 5 µg/mL
CREB (bZIP transcription factor) 06-863 1:200
CyPA (Cyclophilin A) 07-313 1:1000
Endophilin B1 AB10555 1:500-1:1000
FUBP3 07-742 2 µg/mL
G9a (BAT8) 09-071 1 µg/mL
GAPDH MAB374 1:8000
HDAC11 09-827 0.5 µg/mL
Hexim 1 07-955 4 µg/mL
HLX1 09-084 2 µg/mL
IGF2 mRNA-binding protein 2 07-103 4 µg/mL
IGF2 mRNA-binding protein 3 07-104 2 µg/mL
IKKa 07-1007 1 µg/mL
IKKb 07-1008 2-10 µg/mL
IRS1, clone 58-10C-31 05-784R 0.1 µg/mL
JunD 07-1334 1:2000
LSM14A ABE37 1 µg/mL
MAP Kinase 1/2 (Erk-1/2) 06-182 1:500
MYPT1, phosphothreonine (Thr696) ABS45 0.1 µg/mL
NES, Nestin AB5922 1:1000
NEFL, 70 kDa clone DA2 MAB1615 1:200
P38/SAP-K2 05-454 1:600
P 53 AB565 1:1000
PLCg-1, phosphotyrosine (Tyr783) 07-2134 1:1000
PLK1, phosphoserine (Ser137) 07-1348 0.5 µg/mL
Post-immunodetection chromogenic staining of blots When weak signals were obtained in lanes 5 & 6, blots were stained with Coomassie blue following either chemiluminescence (left blots) or fluorescence (right blots) detection to verify equal loading and protein transfer of all lanes.
Lane 1: Molecular weight marker Lane 2: A431 Pervanadate (PVD) stimulated Lane 3: A431 control non stimulated
(fresh samples)Lane 4: A431 EGFR stimulated
(fresh samples)Lane 5: A431 control non stimulated
(old samples)Lane 6: A431 EGFR stimulated
(old samples)
1 2 3 4 5 6 1 2 3 4 5 6
Chemiluminescence detection
Blot stained after detection
1 2 3 4 5 6 1 2 3 4 5 6
Fluorescence detection
Blot stained after detection
ORdERINg INfORMATIONDescription Detection Method Qty/Pk Catalogue No.
Bløk-CH Reagent Chemiluminescence detection 500 mL/bottle WBAVDCH01
Bløk-FL Reagent Fluorescence detection 500 mL/bottle WBAVDFL01
Bløk-PO Reagent Phosphoprotein detection 500 mL/bottle WBAVDP001
BlockingAntibody Incubation
Bløk-PO reagent preserves the phosphorylation state of the protein
Chemiluminescence detection of pERK in EGF-stimulated A431 lysate (10-2.5 µg/lane,12-110, Millipore) using anti-pERK antibody (1:200, 05-797R, Millipore). Bands were detected using Luminata Forte Western HRP substrate (WBLUF0500, Millipore). pERK phosphorylated doublet indicated by arrow. NFDM - Non-fat dry milk
Competitor L Competitor P NFDM Bløk – PO
SNAP i.d. PublicationsMihrshahi R, Barclay AN, Brown MH. J Immunol. 2009 Oct 15; 183(8):4879-86.
Fujimori K, Ueno T, et al. J Biol Chem. 2010 Mar 19; 285(12):8880-6.
Sakane A, Honda K, Sasaki T. Mol Cell Biol. 2010 Feb; 30(4):1077-87.
Many more to be found on www.millipore.com/SnapPub.
Validated for the SNAP i.d. system
For a complete listing, visit the SNAP i.d. Antibody Optimization Reference Guide at www.millipore.com/SNAPab.
10 11
The new Luminata Western HRP Substrates
are a family of premixed, ready-to-use
chemiluminescent reagents for the detection
of HRP-based Westerns. Pour directly onto
the Western membrane without worrying
about pipetting error. The Luminata Classico,
Crescendo and Forte substrates cover a broad
range of sensitivities for all detection needs.
Luminata Western HRP SubstratesPremixed for convenience. Formulated for optimal results.
KEy fEATuRES• Conveniently premixed to avoid
cross contamination of reagents
• Broad range of sensitivities to
cover all detection needs
• Consistent results with less
pipetting error
• Stable at 4 °C or room temperature
• Most sensitive detection reagents
• Compatible with PVDF and
nitrocellulose membranes
Luminata substrates provide better protein detection.Dilution series of stimulated A431 lysates (10-0.6 µg) were resolved by SDS-PAGE then transferred on to Immobilon P membrane. Blots were blocked with Bløk-CH reagent and probed with either anti-PP2, anti-STAT1 (05-987, Millipore), or anti-BRCA1 (05-842, Millipore) primary antibody diluted in Bløk-CH reagent. The appropriate secondary antibodies were added to each antibody. Each blot was visualized with the indicated HRP detection reagent. The limit of detection for each reagent is indicated in left column. Blots were processed using the SNAP i.d. protein detection system.
Luminata Classico Luminata Crescendo Luminata Forte
Benefit Premixed reagent for everyday detection needs
Premixed reagent for detection within the low picogram range
Premixed reagent for detection within the femtogram range
detection limit ~ 6 picograms ~1-3 picograms ~ 400 femtograms
Stock solution stability 1 year at 4 °C 1 year at 4 °C 1 year at room temperature
Related Products
Western Blot Enhancing Reagents Avoid repeating your Western blotting experiment. Enhance your signal with the ChemiLucent™ Plus kit or strip
and reprobe your blot with a different antibody using the ReBlot™ Plus or Blot Restore reagents.
Description Qty/Pk Catalogue No.
ChemiLucent Plus Western Blot Enhancing Kit 1 kit 2650
ReBlot Plus Mild Antibody Stripping Solution, 10X 50 mL 2502
ReBlot Plus Strong Antibody Stripping Solution, 10X 50 mL 2504
Blot Restore Membrane Rejuvenation Kit, 10X 1 kit 2520
ORdERINg INfORMATION
Description Qty/Pk Catalogue No.
Luminata Classico Western HRP Substrates 100 mL WBLUC0100
Luminata Classico Western HRP Substrates 500 mL WBLUC0500
Luminata Crescendo Western HRP Substrates 100 mL WBLUR0100
Luminata Crescendo Western HRP Substrates 500 mL WBLUR0500
Luminata Forte Western HRP Substrates 100 mL WBLUF0100
Luminata Forte Western HRP Substrates 500 mL WBLUF0500
Immobilon Western Chemiluminescent HRP Substrate 100 mL WBKLS0100
Immobilon Western Chemiluminescent HRP Substrate 500 mL WBKLS0500
Protein: PP2~ 6 picograms
Luminata Classico Competitor P Competitor G
Protein: STAT1~ 1-3 picograms
Luminata Crescendo Competitor P Competitor B
Luminata Forte Competitor P Competitor GProtein: BRCA1~ 400 femtograms
detection
Detection of GAPDH in 10 µg (Lane 1), 5 µg (Lane 2), 2.5 µg (Lane 3), and 1.2 µg (Lane 4) of A431 lysate. Blots were treated with the indicated detection reagent and exposed to x-ray film for 5 minutes. Luminata Forte substrate is equivalent to Immobilon Western Chemiluminescent HRP substrates.
Luminata Classico Luminata Crescendo Luminata Forte
Validated for the SNAP i.d. system
10 11
The new Luminata Western HRP Substrates
are a family of premixed, ready-to-use
chemiluminescent reagents for the detection
of HRP-based Westerns. Pour directly onto
the Western membrane without worrying
about pipetting error. The Luminata Classico,
Crescendo and Forte substrates cover a broad
range of sensitivities for all detection needs.
Luminata Western HRP SubstratesPremixed for convenience. Formulated for optimal results.
KEy fEATuRES• Conveniently premixed to avoid
cross contamination of reagents
• Broad range of sensitivities to
cover all detection needs
• Consistent results with less
pipetting error
• Stable at 4 °C or room temperature
• Most sensitive detection reagents
• Compatible with PVDF and
nitrocellulose membranes
Luminata substrates provide better protein detection.Dilution series of stimulated A431 lysates (10-0.6 µg) were resolved by SDS-PAGE then transferred on to Immobilon P membrane. Blots were blocked with Bløk-CH reagent and probed with either anti-PP2, anti-STAT1 (05-987, Millipore), or anti-BRCA1 (05-842, Millipore) primary antibody diluted in Bløk-CH reagent. The appropriate secondary antibodies were added to each antibody. Each blot was visualized with the indicated HRP detection reagent. The limit of detection for each reagent is indicated in left column. Blots were processed using the SNAP i.d. protein detection system.
Luminata Classico Luminata Crescendo Luminata Forte
Benefit Premixed reagent for everyday detection needs
Premixed reagent for detection within the low picogram range
Premixed reagent for detection within the femtogram range
detection limit ~ 6 picograms ~1-3 picograms ~ 400 femtograms
Stock solution stability 1 year at 4 °C 1 year at 4 °C 1 year at room temperature
Related Products
Western Blot Enhancing Reagents Avoid repeating your Western blotting experiment. Enhance your signal with the ChemiLucent™ Plus kit or strip
and reprobe your blot with a different antibody using the ReBlot™ Plus or Blot Restore reagents.
Description Qty/Pk Catalogue No.
ChemiLucent Plus Western Blot Enhancing Kit 1 kit 2650
ReBlot Plus Mild Antibody Stripping Solution, 10X 50 mL 2502
ReBlot Plus Strong Antibody Stripping Solution, 10X 50 mL 2504
Blot Restore Membrane Rejuvenation Kit, 10X 1 kit 2520
ORdERINg INfORMATION
Description Qty/Pk Catalogue No.
Luminata Classico Western HRP Substrates 100 mL WBLUC0100
Luminata Classico Western HRP Substrates 500 mL WBLUC0500
Luminata Crescendo Western HRP Substrates 100 mL WBLUR0100
Luminata Crescendo Western HRP Substrates 500 mL WBLUR0500
Luminata Forte Western HRP Substrates 100 mL WBLUF0100
Luminata Forte Western HRP Substrates 500 mL WBLUF0500
Immobilon Western Chemiluminescent HRP Substrate 100 mL WBKLS0100
Immobilon Western Chemiluminescent HRP Substrate 500 mL WBKLS0500
Protein: PP2~ 6 picograms
Luminata Classico Competitor P Competitor G
Protein: STAT1~ 1-3 picograms
Luminata Crescendo Competitor P Competitor B
Luminata Forte Competitor P Competitor GProtein: BRCA1~ 400 femtograms
detection
Detection of GAPDH in 10 µg (Lane 1), 5 µg (Lane 2), 2.5 µg (Lane 3), and 1.2 µg (Lane 4) of A431 lysate. Blots were treated with the indicated detection reagent and exposed to x-ray film for 5 minutes. Luminata Forte substrate is equivalent to Immobilon Western Chemiluminescent HRP substrates.
Luminata Classico Luminata Crescendo Luminata Forte
Validated for the SNAP i.d. system
Millipore, Advancing Life Science Together, SNAP i.d., Immobilon, Amicon, and Ultracel are registered trademarks of Millipore Corporation. ReNcell, PureProteome, Luminata, Bløk, ReBlot, ChemiLucent, and the M mark are trademarks of Millipore Corporation. Qdot is a trademark of Quantum Dot Corporation. Kodak is a registered trademark of Eastman Kodak Company. Lit. No. PB1033EN00 Printed in the USA 03/10 LS-SBU-10-02831 ©2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.
Immobilon Transfer MembranesDescription Size Qty/Pk Catalogue No.
Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850
26.5 cm x 3.75 m 1 roll IPVH00010
Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850
26.5 cm x 3.75 m 1 roll ISEQ00010
Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810
26.5 cm x 3.75 m 1 roll IPFL00010
SNAP i.d. System
Description Components Qty/Pk Catalogue No.
SNAP i.d. Protein Detection System WBAVDBASE
SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01
Double Blot Holder 30/pk WBAVDBH02
Triple Blot Holder 20/pk WBAVDBH03
Antibody Collection Tray 20/pk WBAVDABTR
SNAP i.d. Blot Roller 1/pk WBAVDR0LL
Bløk Noise-Canceling ReagentsDescription Detection Method Qty/Pk Catalogue No.
Bløk-CH Reagent Chemiluminescence Detection 500 mL/bottle WBAVDCH01
Bløk-FL Reagent Fluorescence Detection 500 mL/bottle WBAVDFL01
Bløk-PO Reagent Phosphoprotein Detection 500 mL/bottle WBAVDP001
Luminata Western HRP SubstratesDescription Qty/Pk Catalogue No.
Luminata Classico Western HRP Substrate
500 mL WBLUC0500
Luminata Crescendo Western HRP Substrate
500 mL WBLUR0500
Luminata Forte Western HRP Substrate
500 mL WBLUF0500
Western Blotting Enhancing ReagentsDescription Qty/Pk Catalogue No.
ChemiLucent Plus Western Blot Enhancing Kit
1 kit 2650
ReBlot Plus Mild Antibody Stripping Solution, 10x
50 mL 2502
ReBlot Plus Strong Antibody Stripping Solution, 10x
50 mL 2504
ORdERINg INfORMATION
Western Blotting ToolsPublication Quality Westerns in Minuteswith Millipore’s Pre-optimized Products
TO PLACE AN ORdER In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)
In Europe, please call Customer Service:
France: 0825.045.645 • Spain: 901.516.645 Option 1 • Germany: 01805.045.645 • Italy: 848.845.645 • UK: 0870.900.46.45
For other countries across Europe and the world, please visit www.millipore.com/offices.
For Technical Service, please visit www.millipore.com/techservice.
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