Western Blot Detection Support — Getting Started

iBind™ Western Systems are benchtop devices that automate immunodetection steps. These devices use no external power source, and rely on mechanical pressure to generate sequential lateral flow (SLF) of immunodetection reagents to perform hands-free blocking, antibody binding, and washes for western detection workflows.

Two iBind™ Western Systems are available: containing i) original iBind™ Western Device, which accommodates one mini blot at a time, and ii) the iBind™ Flex Western Device, which accommodates one or two mini blots, one midi blot, or up to six vertically cut membrane strips (for separate antibodies) at a time.

The original iBind™ Western System (Cat. No. SLF1000) consists of:

• iBind™ Western Device
• iBind™ Blotting Roller
• iBind™ Window Cover

You would need to purchase the following items separately:

• iBind™ Cards (Cat. No. SLF1010)
• iBind™ Solution Kit (Cat. No. SLF1020) for preparing blocking, dilution, and washing buffers for chemiluminescent or chromogenic detection
• iBind™ Fluorescent Detection Solution Kit (Cat. No. SLF1019) for preparing blocking, dilution, and washing buffers for fluorescent detection
• Novex™ AP Mouse Chemiluminescent Detection Kit (Cat. No. SLF1021) or Novex™ AP Rabbit Chemiluminescent Detection Kit (Cat. No. SLF1022)

We do not recommend using your own solutions. The iBind™ Solution and iBind™ Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind™ Western device. We cannot guarantee the performance with any other solutions.

Yes, membranes that are smaller than 9 cm x 9 cm can be used with the iBind™ Western device. It is recommended to place the strips or smaller blots centrally. When using larger membranes, care must be taken to ensure that no part of the membrane is in contact with the stack on the iBind™ card.

It is sufficient to block the membrane in iBind™ Solution/ iBind™ Fluorescent Detection (FD) Solution for the time prior to making antibody dilutions. It is not necessary to block the membrane overnight in the blocking solution. The membrane will be subjected to additional blocking in the iBind™ Solution/ iBind™ Fluorescent Detection (FD) Solution before primary antibody reaches the membrane.

• Rinse the iBind™ Western Device under running water after each use and allow the device to dry before additional usage. No use of detergent is recommended as any residual detergent may interfere with results.
• To maximize the life of the springs in the iBind™ Western Device, store the device with latch unlocked, and the lid open as shown on Page 23 of the manual.

If the PVDF membrane has dried up, it is important to pre-activate it with 100% methanol and then rinse in distilled water before immersing it in iBind™ Solution/iBind™ Fluorescent Detection (FD) Solution. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into iBind™ Solution/iBind™ Fluorescent Detection (FD) Solution.

Please use the protocol mentioned on Page 13 of the manual when using the iBind™ Western System in conjunction with the LI-COR™ Odyssey™ Imaging System. We do not recommend using the LI-COR™ solution.

The iBind™ Flex Western System (Cat. No. SLF2000) consists of:

• iBind™ Flex Western Device
• iBind™ Flex Midi Insert
• iBind™ Flex Mini Insert
• iBind™ Flex Multi-Strip Insert
• iBind™ Flex Blotting Roller

You would need to purchase the following items separately:

• iBind™ Flex Cards (Cat. No. SLF2010)
• iBind™ Flex Solution Kit (Cat. No. SLF2020) for preparing blocking, dilution, and washing buffers for chemiluminescent or chromogenic detection
• iBind™ Flex Fluorescent Detection Solution Kit (Cat. No. SLF2019) for preparing blocking, dilution, and washing buffers for fluorescent detection
• Novex™ AP Mouse Chemiluminescent Detection Kit (Cat. No. SLF1021) or Novex™ AP Rabbit Chemiluminescent Detection Kit (Cat. No. SLF1022)

We do not recommend using your own solutions. The iBind™ Flex Solution and iBind™ Flex Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind™ Flex Western device. We cannot guarantee the performance with any other solutions.

It is sufficient to block the membrane in iBind™ Flex Solution/ iBind™ Flex Fluorescent Detection (FD) Solution for the time prior to making antibody dilutions. It is not necessary to block the membrane overnight in the blocking solution. The membrane will be subjected to additional blocking in the iBind™ Solution/ iBind™ Flex Fluorescent Detection (FD) Solution before primary antibody reaches the membrane.

• Rinse the iBind™ Flex well inserts under running water after each use and allow the well inserts to dry before additional usage.
• Handle well inserts with care.
• Store unused well inserts in the drawer in the iBind™ Flex Western Device.
• To maximize the life of the springs in iBind™ Flex Western Device, store the device with latch unlocked, and the lid open as shown on Page 28 of the manual.

If the PVDF membrane has dried up, it is important to pre-activate it with 100% methanol and then rinse in distilled water before immersing it in iBind™ Flex Solution/iBind™ Flex Fluorescent Detection (FD) Solution. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into iBind™ Flex Solution/iBind™ Flex Fluorescent Detection (FD) Solution.

We offer the iBind™ Flex Solution kit and the iBind™ Flex Fluorescent Detection (FD) Solution kit. For standard chemiluminescent or chromogenic detection, we recommend the iBind™ Flex Solution kit (Cat. No. SLF2020) and for fluorescent detection, we recommend the iBind™ Flex Fluorescent Detection (FD) Solution kit (Cat. No. SLF2019).

Please use the protocol mentioned on Page 14 of the manual when using the iBind™ Flex Western System in conjunction with the LI-COR™ Odyssey™ Imaging System. We do not recommend using the LI-COR™ solution.

Yes. Whether using the Mini or Multi-Strip Inserts, empty wells are still in contact with the card and should be filled with water to ensure proper flow of all solutions across the card.

Yes, to run a single mini blot or up to three vertically cut strip blots. However, when using the Mini or Multi-Strip Inserts, wells not in contact with the card should remain empty.

It is not recommended due to the potential for non-uniform flow from top to bottom which could result in a gradient signal intensity across horizontally cut strip blots.

The formulations are identical for both the standard and FD solutions. The only difference is the amount supplied, so the iBind™ Flex Solution Kits are sufficient for 10 midi, 20 mini, or 60 vertically cut strip blots.

Besides being ~1.7 times as wide, the iBind™ Flex Card also includes alignment lines printed on the back that appear when solution is added to the card to help in proper horizontal placement of multiple mini or vertically cut strip blots.

Yes. Due to an intuitive magnetic design, it’s very simple for the user to change the well inserts. However, we highly recommend the user handle the well inserts with care (as engraved on each insert) when changing or washing, and store the other two well inserts in the convenient underside tray when not in use.

If the PVDF membrane has dried up, it is important to pre-activate it with 100% methanol and then rinse in distilled water before immersing it in iBind™ Solution/iBind™ Fluorescent Detection (FD) Solution. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into iBind™ Solution/iBind™ Fluorescent Detection (FD) Solution.

We offer the iBind™ Solution kit and the iBind™ Fluorescent Detection (FD) Solution kit. For standard chemiluminescent or chromogenic detection, we recommend the iBind™ Solution kit (Cat. No. SLF1020) and for fluorescent detection, we recommend the iBind™ Fluorescent Detection (FD) Solution kit (Cat. No. SLF1019).

We offer the following three types of WesternBreeze™ Chromogenic Detection Kits:

• WesternBreeze™ Chromogenic Detection Kit: Anti-Mouse, Cat. No. WB7103
• WesternBreeze™ Chromogenic Detection Kit: Anti-Rabbit, Cat. No. WB7105
• WesternBreeze™ Chromogenic Detection Kit: Anti-Goat, Cat. No. WB7107

Yes, you can purchase it as a standalone (Cat. No. WP20001).

Novex™ AP Chromogenic Substrate (BCIP/NBT) and Novex™ HRP Chromogenic Substrate (TMB) are ready-to-use, single component substrates for sensitive immunodetection of alkaline phosphatase (AP) or horse radish peroxidase (HRP), respectively, on western blots or dot blots. The sensitivity of the AP substrate is in the 100 pg range, while the HRP substrate is in the 1 ng range.*

Novex™ AP Chromogenic Substrate consists of a ready-to-use solution of 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP) and nitroblue tetrazolium (NBT) which forms a black-purple precipitate upon reaction with alkaline phosphatase. It is available as a standalone product (Cat. Nos. WP20001) and is also a component of the WesternBreeze™ Chromogenic Detection Kits.

Novex™ HRP Chromogenic Substrate consists of a ready-to-use non-toxic solution of 3,3’,5,5’-tetramethylbenzidine (TMB) which forms a blue precipitate upon reaction with horse radish peroxidase. It is available as a standalone product (Cat. No. WP20004).

*Note: Sensitivity may vary depending upon the antigen and antibody used for detection.

For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze™ Chromogenic Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze™ Chromogenic Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze ™ Chromogenic Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze™ Chromogenic Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze™ Chromogenic Immunodetection protocol.

Yes, you may purchase them as standalone products using the Cat. Nos. listed below:

• Cat. No. WB7003 (Antibody Wash)
• Cat. No. WB7001 (Blocker/Diluent A)
• Cat. No. WB7002 (Blocker/Diluent B)
• Cat. No. WB7050 (Combo pack containing Blocker/Diluents A & B)

The goat anti-mouse and goat anti-rabbit secondary antibody solutions AP-conjugated are available as standalone products (Cat. Nos. WP20006 and WP20007) but the rabbit anti-goat secondary antibody solution AP-conjugated is not available as a standalone product.

For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze™ Chemiluminescent Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze™ Chemiluminescent Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze ™ Chemiluminescent Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze™ Chemiluminescent Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze™ Chemiluminescent Immunodetection protocol.

Yes, you can purchase them as standalone products and below are the Cat. Nos.:

• Chemiluminescent Substrate, Cat. No. WP20002
• Chemiluminescent Substrate Enhancer, Cat. No. WP20003

Novex™ AP Chemiluminescent Substrate (CDP-Star™) and Novex™ ECL Chemiluminescent Substrate are non-radioactive substrates for chemiluminescence-based immunodetection of alkaline phosphatase (AP) or horse radish peroxidase (HRP, respectively, on western blots or dot blots. Novex™ Chemiluminescent Substrates provide detection sensitivities superior to that of precipitating chromogenic substrates. Low picogram levels of detection can be achieved using either X-ray film or imaging equipment.

Novex™ AP Chemiluminescent Substrate consists of a ready-to-use solution of CDP-Star™, a dioxetane-based substrate for detection of alkaline phosphatase. The Novex™ AP Chemiluminescent Substrate Enhancer consists of a 20X solution of Nitro-Block-II™ that is mixed with the AP Chemiluminescent Substrate when probing blots on nitrocellulose membranes. Do not use the enhancer with PVDF membranes, as it can produce high background. Light emission for this substrate builds rapidly in the first 20 mins after membrane incubation and reaches peak intensity after 45–60 mins. Light emission has a maximal wavelength at 461–466 nm, and continues for several hours and in some cases, for days. Novex™ AP Chemiluminescent Substrate and Novex™ AP Chemiluminescent Substrate Enhancer are available as standalone products (Cat. Nos. WP20002 and WP20003) and are also components of the WesternBreeze™ Chemiluminescent Detection kits.

Novex™ ECL Chemiluminescent Substrate Reagent Kit is a two-part reagent consisting of a luminol and an enhancer for the detection of horse radish peroxidase. Reagent A and Reagent B are mixed in equal volumes before application to the blot. Light emission for Novex™ ECL Chemiluminescent Substrate is most intense from 5–30 minutes after membrane incubation and decreases slowly with time over the course of several hours.

Yes, you may purchase them as standalone products using the Cat. Nos. listed below:

• Cat. No. WB7001 (Blocker/Diluent A)
• Cat. No. WB7002 (Blocker/Diluent B)
• Cat. No. WB7050 (Combo pack containing Blocker/Diluents A & B)
• Cat. No. WB7003 (Antibody Wash)

The goat anti-mouse and goat anti-rabbit secondary antibody solutions AP-conjugated are available as standalone products (Cat. Nos. WP20006 and WP20007) but the rabbit anti-goat secondary antibody solution AP-conjugated is not available as a standalone product.

No, these SuperSignal™ Substrates require different dilutions. If you follow the SuperSignal™ West Pico Chemiluminescent Substrate protocol when you use SuperSignal™ West Dura Chemiluminescent Substrate, you will see high background or a decreased signal intensity and or duration. You must use less (more dilute) primary and secondary antibodies with SuperSignal West Dura Chemiluminescent Substrate (see product instructions for recommended ranges).

SuperSignal™ West Dura Chemiluminescent Substrate is about 10-fold more sensitive than GE Healthcare ECL Substrate or Thermo Scientific™ ECL Substrate (Cat. No. 32106). SuperSignal™ West Dura Chemiluminescent Substrate is also significantly more sensitive than available chemiluminescent substrates for alkaline phosphatase.

Yes and no. Milk contains variable amounts of biotin so it should not be used with avidin/biotin detection systems. Milk also contains varying amounts of phosphoproteins that may make interfere with anti-phosphotyrosine procedures. A variety of blocking buffers are compatible with the substrate.

Background is a relative phenomenon—no blocker will prevent all interactions 100% of the time. While a particular blocking agent may give a perfect signal-to-noise ratio for one set of reaction conditions, it may not work as well for another set of similar conditions. The key is to optimize the system by trying various blocking conditions.

Yes. Avoid using sodium azide during and after probing steps involving horseradish peroxidase (HRP), as this will inhibit HRP activity. Sulfide, cyanide, fluoride and superoxide ions also inhibit HRP to some extent.

The lower detection limit of SuperSignal West Dura Chemiluminescent Substrate is mid-femtogram (1 x 10e–15).

Although blots detected with chemiluminescent substrates can be stripped and reprobed, some antigen/antibody systems are sensitive to the stripping procedure and might not yield the same quality of results on a stripped blot compared to a new blot. Only actual experimentation will yield information as to whether a given system will allow reprobing.

Please see the protocol mentioned in Tech Tip # 23: Strip and reprobe western blots.

Both nitrocellulose and PVDF membranes work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate.

No, these SuperSignal™ Substrates require different dilutions. If you follow the SuperSignal™ West Pico Chemiluminescent Substrate protocol when you use SuperSignal West Femto Chemiluminescent Substrate, you will see high background or a decreased signal intensity and or duration. You must use less (more dilute) primary and secondary antibodies with SuperSignal™ West Femto Chemiluminescent Substrate (see product instructions for recommended ranges).

SuperSignal™ West Femto Chemiluminescent Substrate is about 25- to 50-fold more sensitive than GE Healthcare ECL Substrate or Thermo Scientific™ ECL Substrate (Cat. No. 32106). SuperSignal™ West Femto Chemiluminescent Substrate is also significantly more sensitive than available chemiluminescent substrates for alkaline phosphatase.

The lower detection limit of SuperSignal™ West Femto Chemiluminescent Substrate is low-femtogram (1 x 10e–15).

No, SuperSignal™ Substrates require different dilutions and 5-minute incubation on the blot. You must use less (more dilute) primary and secondary antibodies with SuperSignal™ West Pico Chemiluminescent Substrate (see product instructions for recommended ranges). Please see Tech Tip # 21: Convert to SuperSignal West Pico Chemiluminescent Substrate from ECL Substrate.

When conditions are optimized, SuperSignal™ West Pico Chemiluminescent Substrate is about two times more sensitive than GE Healthcare ECL Substrate or Thermo Scientific™ ECL Substrate (Cat. No. 32106).

The lower detection limit of SuperSignal™ West Pico Chemiluminescent Substrate is low-picogram (1 x 10e–12).

Yes. However, SuperSignal Chemiluminescent Substrates were optimized for use in Western blots. For Southern and Northern blotting, use our North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Cat. No. 17097), North2South Chemiluminescent Substrate for HRP (Cat. No. 17295) or the Chemiluminescent Nucleic Acid Detection Kit, if probing for biotinylated probes (Cat. No. 89880).

The Western Blot Signal Enhancer should be used after transferring proteins to the membrane and before blocking or staining the membrane.

The Western Blot Signal Enhancer relaxes the proteins, allowing the antibody to bind to the protein more efficiently.

The Western Blot Signal Enhancer will work for any chemiluminescent or colorimetric substrate.

We recommend using the Western Blot Signal Enhancer prior to staining and blocking.

After stripping the blot, blocking reagent remains on the membrane so you will get inconsistent results. Therefore, we recommend using the Western Blot Signal Enhancer only before blocking and the first probing of the membrane.

The Western Blot Signal Enhancer does not bind to the membrane or proteins so it should not interfere with an antibody in binding the target protein.

The Miser Antibody Extender Solution should be used after transferring the protein to the membrane and before blocking the membrane.

The Miser Antibody Extender solution gives consistent, reproducible results on nitrocellulose membranes; results can vary with PVDF membrane.

The Miser Antibody Extender Solution should be used after staining with Ponceau S.

The proteins on the blot become more accessible to the antibodies, making it possible for a signal to be produced using less antibody.

If you are happy with your western signal, you can use the Miser Antibody Extender Solution NC to minimize the amount of antibody you need to use and therefore reduce your costs. If you need to increase sensitivity, then you can use the Western Blot Signal Enhancer.

This assay uses a HRP conjugate or AP conjugate that binds to native IgG from various host species, allowing clear, specific western blot detection from IP experiments without interference from denatured IgG. It is used simply as a substitute for secondary antibodies.

The reagent binds to IgG from a wide range of species: bovine IgG2, goat IgG2, human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, rat IgG2c, sheep IgG2, pig, dog, and cat. It will not bind to rat IgG2a and IgG2b or mouse IgG1. If in doubt whether this detection reagent will bind to a specific antibody, perform a dot-blot analysis before the experiment.

The Clean-Blot™ IP Detection is compatible with bovine serum albumin, SuperBlock™ and StartingBlock™ Blocking Buffers and 5% nonfat milk. Verify compatibility with other blocking buffers by dot-blot analysis.

For best results, use the Clean-Blot™ IP Detection with SuperSignal™ Substrates or ECL Western Blotting Substrate. When using other HRP or AP substrates, empirically determine the optimal concentration of the Clean-Blot™ IP Detection Reagent.

Yes. When using a chemiluminescent substrate, membranes can be stripped and reprobed similar to probing with secondary antibodies.

Yes, the iBlot™ Western Detection Kits use the iBlot™ System (original iBlot™ Dry Blotting System or iBlot™ 2 Dry Blotting System) to produce an electrical field that accelerates interactions between antibodies and blocking reagents with membrane-bound antigens. iBlot™ Western Detection Kits require an iBlot™ Dry Blotting System with the P9 program (available in software versions 2.9.5 and higher). If you have the original iBlot™ Dry Blotting System, the firmware is freely available for download at the Instrument Registration page.

We offer iBlot™ Western Detection kits for chromogenic detection and they are available with either anti-mouse or anti-rabbit secondary antibodies. The kits contain the reagents and detection stacks needed for western detection; all you have to supply is the primary antibody. The iBlot™ Western Detection kits for chemiluminescent detection have been discontinued.

iBlot™ Western Detection Stacks come in two sizes: mini and regular; they are supplied with assay spacers to create partitions for analysis using more than one set of antibodies. With the mini size, you can perform detection on one mini-sized membrane or two halves of a mini-sized membrane. With the regular size, you can perform detection on two mini-sized membranes or 4 quarters of a regular-sized membrane (4 x 8 cm).

No. The iBlot™ Western detection stacks have a finite amount of ions that are depleted after a single run, which includes the three detection steps: blocking, primary antibody, and secondary antibody.

No. A single set of stacks is used for all three steps of the western detection.

No, it does not matter. iBlot™ Western Detection Kits are compatible with western blots created using wet or semi-dry methods. You can use one regular-sized membrane (13.5 cm x 8 cm) or 1–2 mini-sized membranes (8 x 8 cm).

Yes. iBlot™ Western Detection Kits are supplied with reusable spacers, specifically for this purpose. Use spacers to separate membrane sections for detection with different antibody solutions at the same time. A detailed protocol is provided in the user manual.

You can use nitrocellulose or PVDF membranes with the iBlot™ Western Detection Kits. However, we highly recommend using nitrocellulose rather than PVDF because less background is seen with nitrocellulose membranes. This results in more sensitive detection.

Yes, as long as you activate the membrane before you start the detection procedure. Make sure that nitrocellulose membranes is wetted completely using water. Activate PVDF membranes by immersing in 100% methanol for several seconds until the membrane is completely wet, then rinse with water. Then proceed with the iBlot™ Western Detection Kit.

The substrate used for the iBlot™ Western Detection Kits reacts with alkaline phosphatase (AP). Therefore, horseradish peroxidase (HRP) substrates will not work for these kits. Other conjugates and substrates of AP also cannot be used with iBlot™ Western Detection Kits. The reagents supplied with the kits have been optimized to obtain the best results.

Native antibodies typically have some negative charge at neutral pH, which allows their electrophoretic migration in low voltage from the carrier matrix onto the membrane. The force of the electric field is counterbalanced by their affinity for antigen, so that binding antibodies are “captured” while unbound antibodies flow through the blocked membrane and onto the bottom stack. Basically, the iBlot™ system electrophoretically focuses antibodies to greatly accelerate their interaction rate with immobilized antigens.

The iBlot™ Western Detection stacks are compatible with LI-COR™ detection if you use a fluorescently-labeled antibody of your own.

No. The primary antibody solution cannot be reused after it has been applied to the matrix.

Use twice the primary antibody concentration used in typical western blot detection procedures. Note that the overall amount of the primary antibody used is the same as in typical traditional methods, because only half the volume of primary antibody solution is needed with the iBlot™ Western detection system.

The shelf life is 6 months, at room temperature.

The shelf life is 12 months, at 4 degrees C.

Yes. To use your reagents with new stacks, you can purchase the stacks alone: iBlot™ Western Detection Stacks, regular size (Cat. No. IB701001) and iBlot™ Western Detection Stacks, mini size (Cat. No. IB701002).

WesternDot™ reagents are whole IgG (H+L) conjugates of our popular secondary antibodies and select monoclonal primary antibodies, or streptavidin conjugates of our red and far-red fluorescent Qdot™ probes, Qdot™ 585, Qdot™ 625, Qdot™ 655 and Qdot™ 800, for western blotting applications. Qdot™ probes have narrow and symmetrical emissions, minimizing overlap with other emission colors, producing less bleed through into adjacent detection channels and allowing multiple colors to be used simultaneously. They are equally optimally excited with any UV or blue light sources commonly available on gel and blot imagers. Furthermore, Qdot™ probes are extremely photostable, making it possible to take multiple images and store dried blots for months with minimal loss of fluorescent signal.

For more information on our Qdot™ technology, see the following links:

http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/Qdot-nanocrystal-technology.html

http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/Qdot/technology-overview.html

WesternDot™ detection has a number of advantages over enzyme-based chemiluminescent or chromogenic detection methods:

• Ability to do multiplex detection on the same blot at the same time
• Simple protocol that is not time-sensitive and does not require mixing of reagents
• Reliable protocol that can never give over- or under-developed blots
• High photostability enabling dried blots to be archived
• Uses more commonly available UV or blue light imagers rather than more-expensive chemiluminescent imagers or film and chemicals
• Sharper bands due to the direct linking of WesternDot™ reagents to the primary antibody target protein, rather than the diffuse edges around a protein band seen with enzyme-based detection methods

Yes, it can be purchased as Qdot™ 625 Streptavidin Conjugate (Cat. No. Q22063, 50 μL or Cat. No. A10196, 200 µL).

Yes, they can be purchased as Cat. Nos. B2763 and B2770, respectively.

All standalone WesternDot™ reagents are provided with sufficient material to stain 25 Mini gel blots. The WesternDot™625 Goat anti-mouse and goat anti-rabbit kits (Cat. Nos. W10132 and W10142) are provided with sufficient reagents to stain 20 Mini gel blots.

The WesternDot™ detection reagents are stable for at least 6 months from the date of receipt when stored at 2–8 degrees C, We do not recommend freezing them.

Both nitrocellulose and PVDF membranes can be used. For better results, we recommend using nitrocellulose membrane to minimize background from membrane autofluorescence.

For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternDot™ immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternDot™ immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot™ immunodetection protocol.

For Native-PAGE Western blots, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 minutes before proceeding to the WesternDot™ immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot™ immunodetection protocol.

No single buffer formulation works best for every protein/antibody. 2% casein, 5% non-fat dry milk, or 1/2X fish serum (e.g., SEA BLOCK) in 1x PBS/TBS work well for most target protein and antibody combinations. Do not use BSA-containing solutions for blocking or incubating WesternDot™ conjugates as their use may cause high background and/or reduced signal. For primary antibodies that are incompatible with casein or milk (e.g., many antiphosphoprotein antibodies), use fish serum or a 0.5% BSA-containing solution. If using BSA, use for the primary antibody incubation only; in all other steps, use 2% casein, 5% non-fat milk or 1/2x fish serum.

The ideal working concentration can vary between different primary antibodies. For best results, one should refer to the antibody vendor’s suggested conditions, or optimize the working concentration for a primary antibody prior to use.

The recommended WesternDot™ secondary antibody conjugate working dilution is 1:500. Each vial of WesternDot™ secondary antibody conjugate contains sufficient material for twenty-five western blots at this working dilution.

For best results, refer to the vendor’s suggested incubation time, or optimize incubation time. Generally, the primary incubation is carried out for one hour at room temperature, or overnight at 4 degrees C. We recommend an incubation time of 1 hour at room temperature for WesternDot™ secondary antibody conjugates.

Tween™ 20 is a detergent commonly used in western blot buffer formulations. For WesternDot™ secondary conjugates, we found the use of Tween™ 20 in buffers to be suitable for nitrocellulose membrane blots, but not necessary for desirable results. Because Tween™ 20 leads to high background on PVDF membrane it is not recommended for use with WesternDot™ secondary conjugates in combination with PVDF. If PVDF membranes are used, Tween™ 20 should be omitted from all the buffer formulations.

No, it is not necessary. Qdot™ probes are photostable under normal room light illumination, so blots can be incubated and dried without covering to protect from light.

No, the WesternDot™ staining solution should be used as soon as it is prepared and can only be used once. Qdot™ probes are not stable long-term diluted in buffer.

The process for multiplexed protein detection is the same as for single protein detection. After transferring proteins onto PVDF or nitrocellulose membranes and blocking, incubate the blot in a mixture of primary antibody solution for each protein target followed by detection with a mixture of WesternDot™ secondary antibody conjugates against each primary antibody. For example, to do a three-color multiplexing of Akt, phospho-Akt and GAPDH, incubate the blot in a mixture solution of rabbit anti Akt antibody, mouse anti phospho-Akt and chicken anti GAPDH, followed by detection with a mixture of WesternDot™ 605 goat anti rabbit IgG, WesternDot™ 655 goat anti chicken whole IgG and WesternDot™ 800 goat anti mouse IgG.

A western blot containing serial dilutions (20–3 μg protein) of lysates from unstimulated (lanes 2–5) and hEGF-stimulated (lanes 6–9) A431 cell lysate was probed with mouse anti-EGFR, rabbit anti–phospho-EGFR, and chicken anti- GAPDH antibodies, followed by WesternDot™ 800 goat anti-mouse (blue), WesternDot™ 585 goat anti-rabbit (red), and WesternDot™ 655 goat anti-chicken (green) conjugates. The merged image shows overlaid red and blue bands as purple. The blot contains MagicMark™ XP Western Protein Standard (lane 1, Cat. No. LC5603) and was imaged using the Fujifilm™ LAS-4000 gel imager.

Yes, blots can be stripped and reprobed with WesternDot™ reagents. We found that in general, the WesternDot™ conjugate was removed easier than standard dye or protein-labeled secondary conjugates and that the primary antibody was the hardest to remove. You should be able to use any stripping solution that is strong enough to remove the primary antibody. We have obtained good results with Restore™ and Restore™ Plus stripping buffers following their protocol at room temperature. It may take heating at 50 degrees C to fully remove the primary well. The multiplexing capability of WesternDot™ reagents enables simultaneous detection of multiple proteins using appropriately compatible primary antibodies and WesternDot™ reagents, so that stripping and reprobing is no longer required.

Yes, it is possible to multiplex detection with WesternDot™ reagents and WesternBreeze™ CDP-Star™, WesternBreeze™ BCIP/NBT and ECL reagents. WesternBreeze™ CDP-Star™ is the preferred detection reagent for this application. Simply co-incubate in the primary antibodies and then in the secondary antibodies following the WesternDot™ detection protocol. The CDP-Star™ chemiluminescent signal is not detected with UV excitation and emission filters used for WesternDot™ imaging, so the WesternDot™ signal can be imaged before CDP-Star™ substrate incubation or afterwards on wet or dried blots. WesternBreeze™ BCIP/NBT and ECL signals will be detected in the WesternDot™ image, so the WesternDot™ signal must be imaged first on a wet blot before addition of these detection reagents. Here is a link to a more detailed protocol.

Yes, western blot processing instruments work well with WesternDot™ reagents.

Any basic gel or blot imager with UV or blue light excitation and an ethidium bromide filter can be used with for single-color detection of WesternDot™ 625 conjugates. The E-Gel™ imager with the UV or blue light base and the orange filter can be used for detecting single color WesternDot™ 585 or WesternDot™ 625 conjugate-stained blots. For two or three-color multiplexing, a GE Healthcare ImageQuant™ LAS 4000 includes filter sets suitable for imaging WesternDot™ conjugates in the 585, 625, 655, and 800 colors. Other specific imager and filter combinations for all the WesternDot™ reagents can be found in Table 2 of the Western Dot™ Secondary Antibody Conjugates manual.

The photostability of WesternDot™ reagents enables taking multiple images without loss of signal intensity.

MagicMark™ protein standard (Cat. No LC5602) labeled with WesternDot™ 655 goat anti-mouse secondary antibody conjugate; left panel is the first image and right panel is the 10th image.

The photostability of WesternDot™ reagents enables long-term storage of dried blots without loss of signal intensity.

A nitrocellulose blot probed for β-tubulin and detected with WesternDot™ 655 goat anti-mouse reagent; left image is the initial image and right image is after 3 months storage at ambient temperature showing no loss in signal.

No, blots can be imaged wet or dried. For long-term stability of signal, blots should be kept dry.

The brightest signal and lowest sensitivity are obtained when exciting in the UV range, but imagers equipped with a blue light source (e.g., 450 nm or 473/488 nm) can also be used.

When imaging a WesternDot™ conjugate-stained blot, the blot should be placed with the protein side toward the light source. On a UV transilluminator, this means that the blot should be placed face down.

No, the Li-COR™ Odyssey imagers use excitation wavelengths of 685 nm and 785 nm. For optimal sensitivity, you need to use an instrument that has UV or blue light excitation. WesternDot™ reagents have weak excitation near their emission peak, so you may see some signal, but sensitivity will be much lower than for far-red fluorescent dyes.

This is not recommended, as Qdot™ probes used to make the WesternDot™ conjugates are significantly larger than IgGs. Qdot™ probes are estimated to be about 1.5–2 million Da (similar in size to an IgM), so permeability through the gel matrix is going to be much lower than for an unlabeled antibody or even an HRP- or alkaline phosphatase-labeled antibody. It may be possible in a very low percent acrylamide gel with some optimization, but sensitivity will be less than on a membrane.

The sensitivity of WesternDot™ detection reagents and the far-red dyes are all similar to or better than ECL detection. Both types of reagents use similar staining protocols and can be archived on dried blots. Also, WesternDot™ reagents and far-red dyes are both directly linked to the primary antibody target protein, so the resolution of the stained protein band is very sharp, rather than the diffuse edges around a protein band that is seen with chemiluminescent and chromogenic detection via an enzyme/substrate. The advantage of the WesternDot™ reagents over far-red fluorescent dyes is that UV-blue excitation wavelengths are so far removed from the emission wavelengths that there is virtually no autofluorescence background from the blot membrane or blocking proteins, giving much better signal to noise and thus very high contrast images. Far-red fluorescent dyes are excited close to their emission wavelengths, so there will be some autofluorescence background from the blot membrane and blocking proteins. WesternDot™ detection reagents are much more photostable than fluorescent dyes and do not require protection from light during staining or handling. WesternDot™ detection reagents require a UV or blue light excitation source commonly available on most gel and blot imagers and do not require the purchase of a specialized imager. WesternDot™ 625 reagents use the same excitation sources and emission filters as ethidium bromide-stained gels.

This may be possible, but has not been validated. A biotinylated probe should be detected with any of our streptavidin labeled Qdot™ probes (such as our Qdot™ 625 streptavidin conjugate, Cat. No. Q22063) using standard northern or Southern blotting protocols.

Detection sensitivity with Alexa Fluor™ 680 and 790 conjugated secondary antibodies is comparable to detection with Li-COR™ IRDye 680 and 800 dyes used at the same concentration.

Alexa Fluor™ 680 and 790 secondary antibody reagents have similar sensitivity as ECL chemiluminescent detection. The sensitivity is in the picogram range. Far-red fluorescent detection has a number of advantages over enzyme-based chemiluminescent or chromogenic detection methods:

• Ability to do multiplex detection on the same blot at the same time
• Simple protocol that is not time-sensitive and does not require mixing of reagents
• Reliable protocol that can never give over- or under-developed blots
• High photostability enabling dried blots to be archived
• Uses more commonly available UV or blue light imagers rather than more-expensive chemiluminescent imagers or film and chemicals
• Sharper bands due to the direct linking of the far-red secondary antibody conjugate to the primary antibody target protein, rather than the diffuse edges around a protein band seen with enzyme-based detection methods

Technically, the non-far red Alexa Fluor™ antibodies could be used on western blots, but they will give very poor sensitivity compared to other detection methods and thus are not recommended. Blot membranes, especially PVDF, have a high fluorescent background, highest in the blue/green range, which increases noise and thus lowers sensitivity. Blocking proteins can also autofluoresce, increasing background. Blot fluorescent background is very low in the far-red range, which is why Alexa Fluor™ 680 and 790 dyes can obtain high sensitivity. A second reason why non-far red Alexa Fluor™ dyes do not make good western blot detection reagents is that the non-Alexa Fluor™ 680 and 790 antibody conjugates are optimized to give high signals for immunofluorescence (IF) and immunohistochemistry (IHC) staining and generally have a high degree of dye:antibody labeling, which can lead to high nonspecific charge-based binding of the dyes to western blotted proteins and the membrane, increasing background staining and thus lowering signal to noise. The Alexa Fluor™ 680 and 790 secondary antibody conjugates have degrees of labeling that are optimized for western detection, so they have a conjugation efficiency that gives a high signal with lower nonspecific binding.

Yes, western blot processing instruments work well with Alexa Fluor™ 680 and 790 labeled secondary antibodies and give similar or better sensitivity compared to manual processing. See Figure 2 in the following link.

A good initial working concentration is ~0.5 µg/mL, which is a 1:4000 dilution of the 2 mg/mL stock. Depending on the abundance of your target, the optimal concentration may be in the range of 0.1–1 µg/mL.

All Alexa Fluor™ 680 and 790 secondary conjugates come supplied as 0.5 mL of a 2 mg/mL stock or as a 1 mg powder, which is sufficient to stain 200 blots at a working concentration of 0.5 µg/mL and 10 mL/blot.

WesternDot™ detection reagents are stable for at least 6 months from the date of receipt when stored at 2–8 degrees C, We do not recommend freezing them.

Both nitrocellulose and PVDF membrane can be used. For better results, we recommend using nitrocellulose membrane to minimize background from autofluorescence.

Yes, Alexa Fluor™ 680 and 790-stained blots can be imaged wet or dried. We recommend drying blots for long-term storage.

After the plates are blocked, remove the blocking buffer and air dry the plates for several hours. Store plates with desiccant in a plastic bag at 4 degrees C. Change desiccant after 24 hours for optimal storage. The plates should be stable for at least 12 months.

No. Unless it includes “T20” in the name, for example, SuperBlock T20 (PBS) Blocking Buffer, which contains 0.05% Tween 20.

No. It is supplied at 1X concentration and should be used “as is”.

Typical procedures use 1 hour at room temperature or overnight at 4 degrees C. However, sufficient blocking in many procedures is possible in 3 x 2 minutes at room temperature.

Its shelf life is one year at 4 degrees C.

Kathon Preservative, a broad spectrum antimicrobial agent, is used at a concentration of 600 ppm.

Allow any colloidal carbon, if present, to settle to the bottom of the bottle and pouring off what you need to use. We do not recommend centrifugation or filtering because these procedures may reduce the product's blocking capabilities.

There are two types of stripping buffers, each available in two different sizes:

• Restore Western Blot Stripping Buffer: Cat. No. 21059 (500 mL), Cat. No. 21063 (5 L) and Cat. No. 21062 (30 mL)
• Restore Plus Western Blot Stripping Buffer: Cat. No. 46430 (500 mL) and Cat. No. 46428 (30 mL)

Restore Western Blot Stripping Buffer gently but effectively removes the primary and secondary antibodies from the membrane to allow re-probing on the same membrane. The buffer is formulated to work for a wide variety of interactions, but there are some high affinity antigen-antibody interactions that require more stringent stripping conditions. Restore Plus Western Blot Stripping Buffer was developed for these difficult to strip interactions.

No. Every binding interaction is slightly different in terms of mode (i.e., actual amino acids or functional groups that interact) and affinity (strength of binding under a given set of buffer conditions). First try Restore Western Blot Stripping Buffer for 15 minutes at room temperature. If antibody removal is incomplete, optimize the stripping conditions by increasing the time and temperature. If this fails to completely strip the antibodies, then switch to the more stringent Restore Plus Western Blot Stripping Buffer.

Yes. Both incubation time and temperature must be optimized for best results.

Yes. The stripping buffers work to separate the antibody from the antigen, so the membrane to which the antigen is bound generally will not affect the stripping.

After stripping with Restore Buffer, re-blocking of the membrane is usually not necessary but may help to decrease background in some situations. By contrast, re-blocking is required after stripping with Restore Plus Buffer.

No. The antibodies are removed but the substrate leaves a permanent precipitate on the membrane that cannot be removed. Restore and Restore Plus Buffers are designed for procedures using chemiluminescent substrates.

No. Restore and Restore Plus Buffers are not stringent enough to break the avidin-biotin interaction. A buffer capable of breaking this interaction would likely also damage the target protein, making it undetectable.

No. Although the fluorescent antibodies, like other antibodies, can be stripped with Restore Buffers, stripped membranes typically produce unacceptable background for subsequent fluorescent detection methods. We recommend Restore Fluorescent Western Blot Stripping Buffer, Cat. No. 62299 (20 mL) and Cat. No. 62300 (100 mL).

Note: Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane (Cat. No. 22860) only.

Wash the membrane with the same buffer as was used between antibody probing steps during the western blotting procedure.

The stability of the attached (transferred and bound) protein will determine the number of times the membrane can be successfully re-probed after stripping. The protein may withstand stripping as many as four times or as few as one time.

Yes, the BenchPro™ 4100 Card Processing Station has been discontinued as of December 31, 2014. All instruments purchased before then will be covered under the manufacturer’s 1 year warranty. We will continue to manufacture and sell the BenchPro™ 4100 consumables until December 31, 2017 to support our customers who have purchased the instrument.

Clean the surface of the BenchPro™ 4100 Card Processing Station with a damp cloth. Do not use harsh detergents or solvents to clean the unit. Rinse the reagent tray assembly and waste basin with water after each use. If bottles and vials are to be reused, clean with mild detergent and rinse thoroughly with ultrapure water. Do not autoclave trays or reagent bottles and vials. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 mL vials to prevent any reagent cross-contamination.

The 25 mL Reagent Vials can be purchased separately as a set of 50 vials/package (Cat. No. WP3001). However, we do not offer the Reagent Bottles as standalone products. You may purchase them from Cole Parmer

250 mL Reagent Bottles (Set 1) (Cat. No. EW-06019-76) (http://www.coleparmer.com/Product/Bottles_Squre_HDPE_250ml_10_pk/EW-06019-76

125 mL Reagent Bottles (Set 2) (Cat. No. EW-06254-20) (http://www.coleparmer.com/Product/Thermo_Scientific_Nalgene_Sterile_PETG_Media_Bottles_125_mL_48_box/EW-06254-20)

We do not offer the 6 ft Drain tubing for Auto Drain. It is ¼” OD x 1/8” ID and you may purchase it from http://www.mcmaster.com/#catalog/119/136/=q5ikda.

The BenchPro™ 4100 Card Processing Station is pre-programmed with three of the most popular western protocols: two for Western Breeze™ immunodetection and one for standard ECL detection. These protocols are programmed to return the primary antibody to the original vial. Additionally, 17 more custom protocols can be stored in the system’s memory. The custom protocols can be programmed to return any of the reagents back to the original container, if desired.

The instrument has the capacity to process 1–4 Western Cards. Each card could process 1 or 2 membranes at a time, placed back to back. Therefore, the system has the capacity to simultaneously process up to 8 membranes in each run.

Yes, as each card is filled from a separate set of vials.

8.5 cm x 8.5 cm is the maximum membrane size that can be processed.

There is no need to use all 4 cards at a time. You can use one card or 2-4 cards at a time.

No, the cards are not meant to be reused. The instrument detects used cards by a color indicator spot at the lower right hand corner of the card that turns red after the card is used.

The operating temperature range is 4–40 degrees C. So the instrument can be operated in the cold room or incubator if needed. However, we have only tested for a few days in the cold room.

Yes, the processor can be programmed to return reagents back into the same vials after use.

We do not have any validated protocols for this, but we suggest covering the Western Card with foil or running the experiment in a dark room if possible.

No, but please ensure that the reagent bottles and vials have been loaded in the same tray row as the cards that will be used. The unit automatically detects which slots contain cards and only the slots containing cards are functional during processing.

The BenchPro™ 4100 Card Processing Station comes with a one (1) year warranty from the date of delivery. The three-year extended warranty (Cat. No. A12463) is no longer available since the device has been discontinued as of December 31, 2014.

The fuse is 250 V, 1.25 A. We do not offer it as a standalone product, but it can be purchased from electronics vendors such as RadioShack.

The faster program will generally give sensitivity in detection that is 70–80% of the standard WesternBreeze™ immunodetection protocol.

About 0.5–1 mL of each buffer will remain in the card and tips after the run.

Unfortunately, the abort does not send the reagents back to where they came from; they randomly drain through the card.

The BenchPro™ 4100 allows for overnight incubation by increasing the time of the primary antibody incubation step to several hundred mins. The rest of the reagents need to be loaded in the instrument from the beginning, though, as there is no way to halt the run the next day, remove the card to load new reagents, then continue the same protocol with the same card as the previous day. If there is concern about reagents sitting overnight at room temperature, the instrument can be run in a cold room or deli cooler.

Functionally, steps and sub-steps will perform the same. However, you cannot program a sub-step without first programming a step (an error message will display if you try). If you select 'step' and then immediately select 'sub-step', the 'step' will default to 1 min for the 'minutes' but no 'times' will be saved and the protocol that is saved will display the entry as a 'step'. This can be seen when the protocol is viewed again after it is saved.