Western Blotting Support — Getting Started

Midi gels can be transferred using:

• iBlot™ Dry Blotting System in conjunction with Transfer Stacks
• Novex™ Semi-Dry Blotter for simultaneous transfer of up to 2 Midi-gels
• Thermo Scientific™ Pierce™ Power Blotter for simultaneous transfer of up to 2 Midi gels
• Thermo Scientific™ Pierce™ G2 Fast Blotter (will be discontinued as soon as we exhaust current inventory).

Yes, Colloidal Blue Stain can be used before western blotting. However, for optimal transfer efficiency, we recommend destaining the gel and then equilibrating in a series of Tris base/Glycine/SDS solutions to increase solubility; when the transfer is complete, the membrane should be treated with methanol to remove the stain prior to chromogenic development (not necessary prior to chemiluminescent detection).

Yes, due to the lack of glutaraldehyde in the sensitizing solution, you should be able to transfer the proteins to a membrane after completely destaining the SilverQuest™ stained gel.

The dye that is bound to the proteins in the pre-stained ladder is charged and covalently bound, so the transfer efficiency of pre-stained ladders is almost always better than that of SDS-denatured proteins. Therefore, pre-stained ladders are not a good measure of transfer success. On the other hand, the Novex™ Reversible Membrane Protein Stain allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. The staining sensitivity is higher than ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.

We recommend cleaning the sponge pads after each use by rinsing with deionized water and squeezing the water out of the sponge pad 3–5 times.

The preferred method of transfer for E-PAGE™ gels is dry blotting using the iBlot™/iBlot™ 2 Dry Blotting System (see Page 27 of the E-PAGE™ Technical Guide). Please note that this works only for E-PAGE™ 48 gels. Semi-dry blotting using the Novex™ Semi-Dry Blotter, Cat. No. SD1000 (Page 33), or semi-wet blotting (Page 37) may also be performed.

NuPAGE™ Transfer Buffer with 10% methanol provides optimal transfer of E-PAGE™ gels in the XCell II™ Blot Module. The NuPAGE™ Antioxidant is used in the transfer buffer for blotting reduced proteins and prevents the proteins from re-oxidizing. The Tris-Glycine Transfer Buffer has not been tested and it is not known as to what the transfer efficiencies would be like.

The iBlot™ Filter Paper is used for blotting Mini or Midi gels. It is placed on top of the gel before placing the Cathode Stack (Top Stack) to protect the gel integrity during the blotting process. The iBlot™ Filter Paper is supplied in two sizes for efficient blotting of mini- and midi-gels.

Note: Failure to use the iBlot™ Filter Paper during blotting of mini- or midi-gels may result in high currents exceeding the current limit leading to an error (Error2) during the run.

We do not recommend using the iBlot™ Filter Paper for blotting E-PAGE™ gels.

To increase efficiency of transfer of high molecular weight proteins from NuPAGE™ gels, we recommend pre-equilibrating the gel in 2x NuPAGE™ Transfer buffer (without methanol) containing 0.02–0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x NuPAGE transfer buffer containing methanol and 0.01% SDS.

Yes, the NuPAGE™ Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.

Yes, we recommend adding the NuPAGE™ Antioxidant to the NuPAGE™ transfer buffer for enhanced blotting results with reduced proteins.

We do not recommend using Carbonate or CAPS transfer buffers to transfer NuPAGE™ gels as the transfer efficiency will be badly compromised. Further, the high pH environment (>pH 9) of these buffers will make the NuPAGE™ Antioxidant non-functional.

We recommend adding 10%methanol to the NuPAGE™ transfer buffer for transfer of one gel and 20% methanol for the transfer of 2 gels.

Chlorobutanol is used as a preservative in the NuPAGE™ transfer buffer and is not necessary for efficient transfer of proteins. You may prepare the buffer without chlorobutanol but keep in mind that the buffer will not be stable for long periods. We recommend using it within 2 weeks.

Tris-Glycine gels may be transferred in CAPS buffer (10mM CAPS (3-cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0.

For blotting Tricine gels, we recommend using 1X Tris-Glycine Transfer Buffer with 20% methanol. The Tris-Glycine Transfer Buffer interferes with protein sequencing. Hence, if you are performing protein sequencing, we recommend using a non-glycine based transfer buffer such as 1X NuPAGE™ Transfer Buffer, 0.5X TBE Transfer Buffer or CAPS buffer (10 mM CAPS (3 cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0).

We recommend using the NuPAGE™ Transfer buffer for blotting of NativePAGE™ gels. PVDF is the recommended blotting membrane and works well in terms of transfer and detection. Nitrocellulose is not compatible with blotting of NativePAGE™ Gels, as the nitrocellulose membrane binds the Coomassie™ G-250 dye very tightly and is not compatible with alcohol-containing solutions needed to destain the membrane and fix the proteins.

For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer.

After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 minutes. Chill the 0.7% acetic acid that will be used as the transfer buffer. Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (–). This is opposite from the typical western blot with SDS, where the negatively charged protein will migrate toward the anode (+) side during the transfer. Transfer for one hour at 10 volts.

Tip for handling the IEF gel: The 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.

Yes, we offer the Mini Blot Module (Cat. No. B1000), designed to be used with the Mini Gel Tank. This blot module will also work with the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014). Please note that the Bolt™ Mini Blot Module (discontinued as of December 31, 2014) is also compatible with both the Bolt™ Mini Gel Tank and the Mini Gel Tank.

Bolt™ Bis-Tris Plus gels can also be transferred using the XCell SureLock™ Mini Cell, iBlot™ Dry transfer system, or using the Novex™ Semi-Dry Blotter.

We recommend rinsing the blot module with deionized water after use. To clean any residual build-up in the blot module, apply 50% nitric acid in deionized water to areas inside the blot module until residual build-up is removed. Once the build-up is removed, rinse the module at least three times in deionized water. Do not submerge the blot module or soak overnight in nitric acid.

Note: Use gloves when preparing the nitric acid solution.

Here are the replacement parts we offer for the Mini Blot Module:

• Gel Knife, Cat. No. EI9010
• Bolt™ Tank Base, Cat. No. B4478640 (Figure 1, below)
• Gel Runner Tank, Cat. No. B4478641 (Figure 2, below)
• Bolt™ Tank Lid, Cat. No. B4478591 (Figure 3, below)
• Cassette Clamp, Right, Cat. No. B4478592 (Figure 4, below)
• Cassette Clamp, Left, Cat. No. B4478593 (Figure 4, below)
• Power Supply Adapters, Cat. No. ZA10001

We recommend using 2 sponge pads to start with. If the sponge pads become thinner with extended use, then an additional sponge pad can be used.

We recommend transferring a single gel per blot module.

Up to two blot modules can be run simultaneously in the Bolt™ Mini Gel Tank to transfer up to two gels per run.

Due to the universal electrode design, the Mini Blot Module fits on either side of the Mini Gel Tank.

The Mini Blot Module is designed exclusively for the Mini Gel Tank. It will also fit in the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014) but will not fit in the XCell SureLock™ Mini Cell or other vendors’ electrophoresis tanks.

Typically, 200–250 mL of 1X transfer buffer is needed per module which is 2–4 times less than with other transfer devices.

No. However, we recommend adding water to the Mini Gel Tank to help dissipate any heat that may be generated during transfer.

We recommend using 1x Bolt™ Transfer buffer with 10% methanol.

We recommend transferring for 60 minutes at 10 volts with nitrocellulose and 20 volts with PVDF. An optional faster transfer for 30–45 minutes at 15 volts can be used, but a slight loss of sensitivity may be observed.

Except for the sponge pads (Cat. No. EI9052), none of the other parts are available separately.

This is perfectly acceptable as the water in the outer buffer chamber only serves as a coolant. We recommend adding deionized water to the outer buffer chamber to avoid exposure of the mini-cell to methanol as the mini-cell is susceptible to methanol.

We recommend rinsing the blot module with deionized water after use. To clean any residual build-up in the blot module, apply 50% nitric acid in deionized water to areas inside the blot module until residual build-up is removed. Once the build-up is removed, rinse the module at least three times in deionized water. Do not submerge the blot module or soak overnight in nitric acid.

Note: Use gloves when preparing the nitric acid solution.

The dimensions of the sponge pads are 8 cm x 9 cm.

No. The solution placed in the outer chamber serves to dissipate the heat generated during blotting. Water is usually used for this purpose. The recommended transfer conditions generate only a minor heat increase, so it is not necessary to run the unit in an ice bucket or to place it in a cold room. However if you are working with very heat-sensitive proteins, you may wish to do so.

For overnight blotting, perform transfer in the cold room with low power to prevent overheating. Transfer at constant voltage of 10–15 V overnight. Depending on the transfer efficiency, adjust the transfer conditions accordingly.

Do not use ethanol or other organic solvents to clean the Novex™ Semi-Dry Blotter. Organic solvents can cause acrylic to crack. Rinse the blotter with deionized water after each use to clean and let air dry. If you must wipe the surface, take particular care not to scratch or otherwise damage the platinum-coated titanium plate.

The Novex™ Semi-Dry Blotter allows simultaneous transfer of 1–2 Midi Gels, 1–4 Mini Gels, or 1–2 E-PAGE™ gels.

For semi-dry western blotting, the transfer buffer must be at twice the concentration used in wet blotting (i.e., 2X) to ensure that there are enough buffering ions present in the smaller volume of liquid.

NuPAGE™ Bis-Tris Gels do not transfer efficiently using the Novex™ Semi-Dry Blotter as compared to blotting with XCell II™ Blot Module. If you decide to use the Novex™ Semi-Dry Blotter for NuPAGE™ -Tris Gels, use the protocol provided below to ensure efficient transfer of proteins.

1. Prepare 100 mL of 2X NuPAGE™ Transfer Buffer from 20X NuPAGE™ Transfer Buffer as follows:
   NuPAGE™ Transfer Buffer (20X) 10.0 mL
   NuPAGE™ Antioxidant (for reduced sample) 0.1 mL
   Methanol 10.0 mL
   Deionized water 79.9 mL
   Total Volume 100 mL
   If you are blotting large proteins, please see the Note below.

2. Soak the filter paper and transfer membrane in the transfer buffer. If you are using Novex™ pre-cut membrane/filter sandwiches, use three filter papers (0.4 mm/filter in thickness) on each side of the gel or membrane. If you are not using the Novex™ pre-cut membrane/filter sandwiches, use two thick filter papers.

3. Equilibrate the gel in the transfer buffer (100 mL for Midi gels and 50 mL for Mini gels) for 10 minutes, on an orbital shaker, to remove salts that may increase conductivity and heat during transfer.

4. Assemble the gel/membrane/filter paper sandwich on top of the anode plate as follows:
   filter paper
   filter paper
   filter paper
   membrane
   gel
   filter paper
   filter paper
   filter paper

5. Perform the transfer at 20 V (constant) for 30–60 minutes.

Note: For transfer of large proteins (>100 kDa), pre-equilibrate the gel in 2X NuPAGE™ Transfer Buffer (without methanol) containing 0.02-0.04% SDS for 10 min before assembling the sandwich.

We recommend using 2X NuPAGE™ Transfer Buffer and performing the transfer at 20 V (constant) for 30–60 minutes.

Yes, we recommend using 2X Tris-Glycine Transfer Buffer containing 10% methanol and performing the transfer at 20 V (constant) for 30–60 minutes.

The preferred method of transfer for E-PAGE™ gels is dry blotting using the iBlot™/iBlot™ 2 Dry Blotting System (see Page 27 of the E-PAGE™ Technical Guide). Please note that this works only for E-PAGE™ 48 gels. Semi-dry blotting using the Novex™ Semi-Dry Blotter, Cat. No. SD1000 (Page 33), or semi-wet blotting (Page 37) may also be performed.

For semi-dry transfer of E-PAGE 48 gels, we recommend using 2X NuPAGE Transfer Buffer containing 15% methanol and for E-PAGE 96 gels, we recommend using 2X NuPAGE Transfer Buffer without methanol. Transfer at 25 V (constant) for 30-60 minutes. Please refer to Page 33 of the E-PAGE™ Technical Guide.

Yes, please follow instructions in the manual for it. 

Here is the transfer protocol optimized for the Bio-Rad Semi-Dry Transfer Unit. NuPAGE™ transfer buffer can be used for transfer of Tris-Glycine gels. 

1. Working transfer buffer: 10% methanol, 1:1,000 Antioxidant in 2X NuPAGE™ transfer buffer (Bis-Tris 50 mM and Bicine 50 mM). If you need to prepare 100 mL of the working buffer from the NuPAGE™ 20X Transfer Buffer (Cat. No. NP0006), mix the following: 10 mL of 20X transfer buffer, 10 mL of MeOH, 100 µL of Antioxidant, 80 mL of DI H2O. 

2. Filter papers: The transfer buffer-soaked filter papers of the sandwich are the only reservoir in the Semi-Dry Transfer Cell. If Invitrogen™ pre-cut membrane/filter sandwiches are used, at least 2 extra filter papers (0.4 mm/filter in thickness) on each side of the gel (or membrane) are required. When assembling one gel/membrane sandwich, presoak 6 Invitrogen™ filter papers (or 2 thicker filter papers) and 1 membrane in working transfer buffer (prepared in step 1) and sandwich them on the top of the anode plate as follows: filter paper--filter paper--filter paper--membrane--gel--filter paper--filter paper--filter paper 

3. Blotting conditions: We found 15 V for 15–30 min is optimal for NuPAGE™ transfer buffer in the Bio-Rad™ Semi-Dry Transfer Cell. Semi-dry transfer units from other manufacturers should be used according to unit's instructions. 

4. For transfer of large proteins (100 kDa or larger), pre-equilibrate the gel with 0.02–0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich. Please note that transferring Tris-Glycine gels using NuPAGE™ transfer buffer in the Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell may be less efficient than using Tris-Glycine transfer buffer (Cat. No. LC3675) in the XCell II™ Blot Module (semi-wet).

The iBlot™ Gel Transfer Device has been discontinued and we have launched the iBlot™ 2 Gel Transfer Device, that is an improved version. The iBlot™ Transfer Stacks, Western Detection Stacks and DNA Transfer Stacks are still available for purchase and are to be used exclusively with the original iBlot™ Gel Transfer Device, and are not compatible with the new iBlot™ 2 Gel Transfer Device. Only iBlot™ 2 consumables are to be used with the iBlot™ 2 Gel Transfer Device.

The iBlot™ Dry Blotting System is compatible for use with Bolt™ Bis-Tris Plus, NuPAGE™ Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE™ gels.

The following iBlot™ Transfer Stacks are available for use with the iBlot™ Gel Transfer Device and are also sold separately:

• iBlot™ Gel Transfer Stacks are used to transfer proteins from gels onto nitrocellulose or PVDF membranes (western blotting). Both nitrocellulose and PVDF transfer stacks are available in mini and regular formats.
• iBlot™ DNA Transfer Stacks are used to transfer DNA from gels onto nylon membranes (Southern blotting).

We recommend storing the iBlot™ Transfer Stacks at room temperature.

The minimum guaranteed shelf life of iBlot™ Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months. The expiration date is printed on the package for each stack.

The PVDF membrane is pre-activated and ready for use without any pretreatment with alcohol.

Yes, the Cat. No. for the iBlot™ lid latch is IB1003 and the Cat. No. for the iBlot™ Electrodes (1 pair) is IB1002. Here are the instructions to replace the electrodes.

The iBlot™ Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.

The iBlot™ Dry Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.

It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.

The plastic in the iBlot™ Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

To maximize the recovery of the copper mesh electrodes used in our iBlot™ Transfer Stacks, we have established an agreement with a recycling center in the United States. In order to prepare the copper electrodes for recycling, please follow the instructions listed here.

No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

In theory, you can replace the membrane provided in your iBlot™ Transfer Stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then, either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that we only support the use of iBlot™ Transfer Stacks when they are used with the provided instructions.

Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from the gel to the membrane. We recommend using P3 program with a transfer time of 8–10 minutes for optimal transfer of these proteins.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE™ Novex™ 3-8% Tris-acetate gels for electrophoresis. For the protocol, please see Transferring Large and Small Proteins Using the iBlot™ Dry Blotting System Application Note.

A conventional stripping protocol using 0.1 M glycine, pH 2, works with polyclonal antibodies.

Yes, please take a look at the Western Blotting NativePAGE™ Novex™ Bis-Tris gels Using the iBlot™ 7-Minute Blotting System Application Note.

The iBlot™ Filter Paper is used for blotting mini- or midi-gels. It is placed on top of the gel before placing the iBlot™ Cathode Stack (Top Stack) to protect the gel integrity during the blotting process. The iBlot™ Filter Paper is supplied in two sizes for efficient blotting of mini- and midi-gels. We do not recommend using the iBlot™ Filter Paper for blotting E-PAGE™ gels.

Note: Failure to use the iBlot™ Filter Paper during blotting of mini- or midi-gels may result in high currents exceeding the current limit leading to an error (Error2) during the run.

The iBlot™ E-PAGE™ Tab is a steel tab used during blotting of E-PAGE™ gels. It is attached to the iBlot™ Cathode Stack, Top and is used to pull the transfer stack assembly towards the blotting surface during the de-bubbling process of E-PAGE™ gels.

The De-bubbling Roller is a stainless steel, aluminum roller designed to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks when blotting E-PAGE™ gels. We recommend using the De-bubbling Roller only for E-PAGE™ gels. Other gel types may stretch and tear if pulled through the roller. The De-bubbling Roller is provided with the iBlot™ Dry Blotting System but not with the iBlot™ 2 Dry Blotting System.

The Blotting Roller is a plastic roller attached to a stainless steel handle. It is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel.

Yes, the iBlot™ Transfer Stacks are compatible with Li-COR™ detection.

Small proteins (under 30 kDa) migrate more rapidly than large ones and hence, need less time to transfer from the matrix of the gel to the membrane. While P3 program for 7 minutes works well with most proteins, less time is needed for the transfer of smaller proteins with the iBlot™ device. We recommend using P3 program for less than 5–6 mins. Please refer to Transferring Large and Small Proteins Using the iBlot™ Dry Blotting System Application Note.

The iBlot™ 2 Dry Blotting System is compatible for use with Bolt™ Bis-Tris, NuPAGE™ Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE™ gels (E-PAGE™ 48 only).

We offer iBlot™ 2 Gel Transfer Stacks with integrated nitrocellulose or PVDF membranes for transferring proteins from gels onto nitrocellulose or PVDF membranes, respectively (western blotting). These stacks can also be purchased separately.

We recommend storing the iBlot™ 2 Transfer Stacks at room temperature.

It is currently 9 months, and the expiration date is printed on the box.

No. The results may be inferior after the expiration date.

The iBlot™ 2 Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.

The PVDF membrane is pre-activated and ready for use without any pre-treatment with alcohol.

Yes, we offer the iBlot™ 2 Electrode Replacement Kit, Cat. No. IB28001, that consists of two electrical contacts, two screws, and a round bumper. Please refer to page 37 in the manual for the instructions.

This is the plastic separator that separates the Top Stack from the Bottom Stack. Right before assembling the stack, this separator needs to be removed from the Top Stack and discarded.

Note: In some instances, the membrane may adhere to the separator. If this is the case, use forceps to remove the membrane and place it on top of the Bottom Stack.

It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible. So the recommendation would be to transfer 2 mini gels using a Regular stack.

To maximize the recovery of the copper mesh electrodes used in our iBlot™ 2 transfer stacks, we have established an agreement with a recycling center in the United States. In order to prepare the copper electrodes for recycling, please follow the instructions listed here.

Yes. However, minor optimizations in the transfer protocol (i.e., steps, voltage, time) may be needed for some proteins.

Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from the gel to the membrane. We recommend using a transfer time of 8–10 minutes for optimal transfer of these proteins.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE™ 3-8% Tris-acetate gels for electrophoresis. For the protocol, please refer to page 35 in the manual.

Yes. The device allows programming custom methods.

Yes. The device is designed to do so with no impact on performance.

No. They are disposable and should only be used once.

It is possible but the results may be inferior.

The membranes are compatible with all commonly used detection methods such as staining, immunodetection, fluorescence, etc.

No. Maintain the membrane size identical to the transfer stacks. This helps ensure that there is no direct contact between the top and bottom transfer stacks.

No. Do not trim the iBlot™ 2 Transfer Stacks to fit your gel size.

No. Do not use iBlot™ Transfer Stacks in the iBlot™ 2 Gel Transfer Device, and do not mix components between iBlot™ Transfer Stacks and iBlot™ 2 Transfer Stacks.

Yes, all original iBlot™ stacks are still available for purchase. You can find them in the original iBlot™ Gel Transfer Device Reagents and Resources. However, please note that they are not compatible with the iBlot™ 2 Gel transfer Device.

The Blotting Roller is a plastic roller attached to a stainless steel handle. It is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel.

The iBlot™ Filter Paper is used for blotting mini- or midi-gels. It is placed on top of the gel before placing the iBlot™ Cathode Stack, Top to protect the gel integrity during the blotting process. The iBlot™ Filter Paper is supplied in two sizes for efficient blotting of mini- and midi-gels. We do not recommend using the iBlot™ Filter Paper for blotting E-PAGE™ gels.

Note: Failure to use the iBlot™ Filter Paper during blotting of mini- or midi-gels may result in high currents exceeding the current limit leading to a “High Current Error” during the run.

The iBlot™ 2 Absorbent Pad absorbs any excess liquid formed on the stacks during blotting and generates even pressure on the stack assembly. It is placed on top of the assembled iBlot™ 2 stack prior to transfer. We recommend discarding the iBlot™ 2 Absorbent Pad after every use.

The Pierce™ Power Blotter (Cat. No. 22834) consists of the Thermo Scientific™ Pierce™ Power Station (Cat. No. 22838) with activated Blotting Software and the Thermo Scientific™ Pierce™ Power Blot Cassette (Cat. No. 22835). It is designed for rapid semi-dry transfer of proteins from polyacrylamide gels to nitrocellulose or PVDF membranes. Traditional western blotting techniques require a transfer of one hour to overnight to achieve good transfer efficiency. When used in conjunction with Thermo Scientific™ Pierce™ 1-Step Transfer Buffer, the Thermo Scientific™ Pierce™ Power Blotter is designed to provide transfer efficiency in 5–10 minutes that is equivalent to, or better than, traditional blotting techniques without the need for gel pre-equilibration. This significant reduction in protein transfer time is accomplished by optimizing the ionic strength of the transfer buffer and increasing the current [amps (A)/cm2] flowing through the transfer stack. The system has been verified to work with commonly used pre-cast and homemade SDS-PAGE gels. The Pierce Power Blotter can also be used for standard semi-dry transfer protocols with Towbin buffer.

When the Pierce™ Power Blotter is used in conjunction with Towbin Transfer Buffer, one to four mini-sized gels or one to two midi-sized gels can be simultaneously transferred in 45–60 minutes. Please note that gels simultaneously transferred must have the same formulation.

Yes. The Pierce™ Power Stain Cassette (Cat. No. 22836) can be purchased and will activate the pre-loaded Thermo Scientific™ Pierce™ Power Stainer software once inserted into your device.

No. During the staining, the proteins are fixed to the gel thus the western blotting results would not be optimal. The unstained gel should be used for western blotting.

Yes. The Thermo Scientific™ Pierce™ Power Station automatically recognizes the cassette that is inserted and initialized the correct software menu system.

No. It is not possible to insert the Pierce™ Power Blot Cassette and access the Power Staining protocol or vice versa.

No. Each cassette can only be used for its intended purpose; Pierce™ Power Blot Cassette for blotting (fast protein transfer) and Pierce™ Power Stain Cassette for protein staining. We have optimized the surface area to yield the best results for each application. The smaller stain cassette works better for staining while the larger blot cassette works better for blotting.

Yes. The Thermo Scientific™ Pierce™ G2 Fast Blotter has been discontinued. The direct replacement for the Thermo Scientific™ Pierce™ G2 Fast Blotter is the Thermo Scientific™ Pierce™ Power Blotter (Cat. No. 22834). The 1-Step Transfer buffer (Cat. No. 84731) is still available.

The Thermo Scientific™ Pierce™ Power Blot Cassette (Cat. No. 22835) is compatible with the Thermo Scientific™ Pierce™ G2 Fast Blotter.

We offer the Thermo Scientific™ Pierce™ G2 Blotter- Power Stainer Upgrade Kit. This kit contains the Thermo Scientific™ Pierce™ Power Stain Cassette and a USB Flash Drive loaded with software that will update the Thermo Scientific™ Pierce™ G2 Fast Blotter Control Unit software and add the staining functions.

We offer the following membranes for western blotting:

Prewet the nitrocellulose membrane in distilled water or in 1X transfer buffer for 5 min with gentle shaking. This is to prevent any dry spots in the membrane that may interfere with transfer.

We recommend rinsing the membrane briefly in water, air drying and store it at room temperature in a ziplock bag. Do not place nitrocellulose in the freezer because it will shatter. Unlike PVDF, nitrocellulose membranes should never be pre-wetted in alcohol as it will cause them to shrivel.

We recommend air drying the PVDF membrane and placing it in an envelope, preferably on top of a supported surface to keep the membrane flat. It can be stored indefinitely at –80 degrees C. Right before probing, we recommend re-wetting the membrane with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with the blocking step.

No we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:

1. Novex™ Reversible Membrane Protein Stain (Cat. No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.
2. Coomassie™ (non-colloidal) staining: Stain in 0.1% Coomassie™ Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at –20 degrees C. Sensitivity is approximately at the 50–100 ng level.
3. SimplyBlue™ SafeStain. There is a protocol included in the SimplyBlue™ SafeStain manual for staining dry PVDF membranes but it is not recommended for wet PVDF membranes or nitrocellulose because of the high background.
4. Amido black: same as Coomassie™ but less sensitive. Please see protocol in the manual.
5. Ponceau S: same as Coomassie™ stain but less sensitive. Please see protocol in the manual.
6. UV transillumination: After blotting, place the membrane on a filter paper and allow to air-dry at room temperature for about 10 minutes. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because the membrane is dry, rewet the membrane.

Our PVDF can tolerate acetonitrile but our nitrocellulose cannot.

Yes, both our PVDF and nitrocellulose membranes are compatible with the Li-COR™ instrument.

Drying the PVDF membrane reduces the background staining that can occur with wet membranes. A dry PVDF membrane is very hydrophobic and doesn't wet well, but the areas where proteins are bound are more easily saturated and stainable. 

This will not work with nitrocellulose membranes, and will only work with PVDF membranes stained for a brief period; staining beyond the recommended time will only increase the background and reduce the detectability.