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:
i) containing the original iBind Western Device, which accommodates one mini blot at a time
ii) containing 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.

We do not recommend using your own solutions. The iBind/iBind Flex Solution and iBind/iBind Flex Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind/iBind Flex Western device, respectively. 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.

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. Due to an intuitive magnetic design, it is 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.

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

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 can purchase it as a standalone (Cat. No. WP20001). It will give a purple chromogenic precipitate.

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

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)

Note: Cat. Nos. WB7001 and WB7002 may be ordered by clicking the Quick Order button located at the top right of any page on our website.

Primary antibody recommended dilution range is 0.2 to 1.0 µg/mL (1:1000 to 1:5000 dilution from a 1 mg/mL stock). Secondary antibody recommended dilution range is 10 to 50 ng/mL (1:20,000 to 1:100,000 dilution from 1 mg/mL stock). Please also see Tech Tip #32 Guide to Enzyme Substrates for Western Blotting .

SuperSignal West Pico PLUS Chemiluminescent Substrate enables detection of low-picogram to high-femtogram amounts of target protein on nitrocellulose or PVDF membrane. Please also see Tech Tip #32 Guide to Enzyme Substrates for Western Blotting .

SuperSignal West Pico PLUS Chemiluminescent Substrate can be used as an everyday substrate. For assays requiring higher sensitivity, we recommend using SuperSignal West Dura Extended Duration Substrate or SuperSignal West Femto Maximum Sensitivity Substrate.

SuperSignal West Dura Extended Duration Substrate is about 10-fold more sensitive than GE Healthcare ECL Substrate or Thermo Scientific ECL Substrate (Cat. No. 32106). SuperSignal West Dura Extended Duration Substrate is also significantly more sensitive than available chemiluminescent substrates for alkaline phosphatase. Please also see Tech Tip #32 Guide to Enzyme Substrates for Western Blotting .

The lower detection limit of SuperSignal West Dura Extended Duration Substrate is mid-femtogram (1 x 10^-15). Please also see Tech Tip #32 Guide to Enzyme Substrates for Western Blotting .

SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate. It is oxidized to an excited state product in the presence of the stable peroxide and horseradish peroxidase. As the product decays to a lower energy state, light is released at 425 nm. The emitted light is then captured on X-ray film or by using a CCD camera.

SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30-60 min, the signal produced with SuperSignal West Dura Extended Duration Substrate is stable for 24 hr, allowing multiple film or camera exposures.

The lower detection limit of SuperSignal West Femto Maximum Sensitivity Substrate is low-femtogram (1 x 10^-15). Please also see Tech Tip #32 Guide to Enzyme Substrates for Western Blotting .

We do not recommend swapping protocols/antibody dilutions between different SuperSignal substrates. For example, if SuperSignal West Femto Maximum Sensitivity Substrate is used with the protocol for SuperSignal West Femto Maximum Sensitivity Substrate, it would result in high background. We also recommend using antibody dilutions as suggested in the manual for the respective SuperSignal substrate.

Yes. The SuperSignal western blot substrates are well suited to most imaging systems including CCD camera units; however, most screen-type phosphorimagers give poor results with the SuperSignal substrates.

SuperSignal West Atto Ultimate Sensitivity Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate that can detect low-femtogram to high-attogram quantities of protein.

We recommend using SuperSignal West Atto Ultimate Sensitivity Substrate for detection of proteins in the following situations:
- For proteins with low concentration, not measurable by BCA or Bradford assays
- For low-abundance phosphorylated or glycosylated proteins
- For low-abundance membrane or nuclear proteins
- For low-abundance proteins being detected with a low-affinity antibody or when the antibody is precious
- When sample source is limited or when multiple conditions or assays are being tested on a single sample

SuperSignal West Atto Ultimate Sensitivity Substrate is compatible with Restore Western Blot Stripping Buffer.

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.

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 SuperSignal Western Blot Enhancer kit contains two reagents:

- Antigen Pretreatment Solution: used after transferring proteins to the membrane and before blocking or staining the membrane
- The Primary Antibody Diluent: used to dilute the primary antibody

The reagents in both kits are different. Western Blot Signal Enhancer (Cat. No. 21050) is a 2-step pre-treatment that occurs before blocking. It basically relaxes the protein on the membrane making the epitope easier to detect. SuperSignal Western Blot Enhancer (Cat. Nos. 46640, 46641) contains a membrane treatment reagent and a primary antibody diluent that increase both signal intensity and sensitivity 3- to 10-fold compared to a detecion performed without it. We have found that SuperSignal Western Blot Enhancer shows much less background while enhancing signal and sensitivity when compared to Western Blot Signal Enhancer.

Not necessarily. The SuperSignal Western Blot Enhancer will work with any chemiluminescent substrate (including SuperSignal Western Blot substrates), as well as colorimetric or fluorometric detection systems.

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.

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 Reagent 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 Reagent 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.

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:

https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/qdot-nanocrystal-technology.html

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

WesternDot reagents have similar or slightly better sensitivity compared to ECL detection. The sensitivity is in the picogram range.

Detection of picogram levels of target protein with WesternDot secondary antibody conjugates. Serial dilutions (2500 pg to 5 pg) of purified AKT1 Recombinant Human Protein (Cat. No. P2999) were run on NuPAGE Novex 4–12% Bis-Tris pre-cast gels (Cat. No. NP0321BOX) and transferred to iBlot nitrocellulose membranes (Cat. No. IB3010-32) using the iBlot Gel Transfer Device (Cat. No. IB1001). The membranes were probed with mouse anti-AKT primary antibody (Cat. No. AHO1112). AKT1 signal was detected with either fluorescent WesternDot 625 goat anti-mouse (Cat. No. W10808, top), or ECL using a horseradish peroxidase goat anti–mouse IgG (bottom). Western blots were imaged using the Fujifilm™ LAS-4000 gel imager. The WesternDot 625 blot was imaged using Epi-UV illumination and an ethidium bromide filter, and the ECL blot was imaged using the chemiluminescence detection settings.

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

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.

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.

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.

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.

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.

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. 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.

Yes, the BenchPro 4100 Card Processing Station has been discontinued as of December 31, 2014. The current BenchPro 4100 Western Cards and Reagent Vials will continue to be available for purchase. We offer the new iBind Western Device as an alternative system for automated western blot processing.

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)

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.

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.

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

The iBright CL1500 Imaging System allows imaging of chemiluminescent Western blots, DNA and RNA gels stained with fluorescent nucleic acid stains, and visible protein gels. The iBright FL1500 Imaging System adds the capability of imaging fluorescent Western blots using multiple dyes.

Smart exposure technology rapidly determines optimal exposure time, which helps minimize the potential for over- or underexposed images, and the need to repeat exposures to get the desired signal. Smart exposure is enabled by built-in algorithms which aim to maximize sensitivity without creating band saturation. Smart exposure time varies depending on the gel imaged and bands displayed.

We recommend taking Ponceau staining images in “Protein gel” mode, with the white screen. It is possible to play with the focus/resolution/sensitivity to get bands as sharp as possible. Better quality pictures are obtained when the membrane is wet.

The Power Blotter System (Cat. No. PB0012) comes with the Power Blotter Station (Cat. No. PB0010) plus Power Blotter Cassette (Cat. No. PB0002). The Power Blotter System can transfer one midi or up to two mini gels at the same time. The Power Blotter XL System (Cat. No. PB0013) on the other hand comes with the Power Blotter Station (Cat. No. PB0010) plus Power Blotter XL Cassette (Cat. No. PB0003). The Power Blotter XL System can transfer up to two midi or four mini gels at the same time.

The Power Blotter Cassette (Cat. No. PB0002) is designed to blot 1-2 mini gels at a time or 1 midi gel and the Power Blotter Cassette XL (Cat. No. PB0003) is designed to blot 1-4 mini gels or 1-2 midi gels at a time. Both cassettes can be used with the Power Blotter. On the other hand, the traditional G2 Fast Blotter is only compatible with the Power Blotter Cassette XL (Cat. No. PB0003); the Power Blotter Cassette (Cat. No. PB0002) will not work with the G2 Fast Blotter.

Yes, but you need to ensure that the total thickness of the filter paper stack on either side of the gel/membrane does not exceed 1.8 mm.

Yes, both PVDF and nitrocellulose membranes are compatible with the Power Blotter System/Power Blotter XL System. You can use 0.2 µm and 0.45 µm pore size membranes. Our Power Blotter Pre-cut Membranes and Filters (PVDF and nitrocellulose) come with 0.45 µm pore size membranes. Our Power Blotter Select Transfer Stacks (PVDF and nitrocellulose) come with 0.2 µm pore size membranes.

Even though the Power Blotter System/Power Blotter XL System does not have the staining program, this can be introduced. Please see the user guide for our Pierce Mini/Midi Gel Power Staining Kit for how to create a Custom Staining Method.

Yes, the Power Blotter System/Power Blotter XL System is an open system and you can use your own transfer buffer. However, keep in mind that your own transfer buffer will result in a prolonged transfer time (~60 min), whereas a fast transfer (5-10 min) can be achieved using the Power Blotter 1-Step Transfer Buffer (5X) or the Pierce 1-Step Transfer Buffer.