XCell SureLock™ Mini-Cell and XCell II™ Blot Module - FAQs

Here are some options for obtaining more efficient transfer for larger proteins:

1) Pre-equilibrate the gel with 0.02 to 0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich.

2) Increase the blotting time incrementally (in 15 min intervals).

3) Add 0.01% or 0.02% SDS to the transfer buffer to help facilitate the migration of the protein out of the gel.

4) Decrease the methanol content in the transfer buffer.

5) Switch to a more appropriate lower-percentage gel. A lower-percentage gel may allow better transfer than a higher-percentage gel.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Build-up can be removed with 50% nitric acid. Make a solution of 50% nitric acid in deionized water and carefully apply it to areas inside the blot module until residual build-up is removed. Do not submerge the blot module or soak overnight. Use gloves when preparing the solution. Afterwards, rinse the module thoroughly at least three times in fresh deionized water. This treatment should not harm the plastic.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The NuPAGE Invitrogen Bis-Tris Gels do not transfer efficiently using a Invitrogen Semi-Dry Blotter as compared to blotting with XCell II Blot Module.
If you decide to use Invitrogen Semi-Dry Blotter for NuPAGE Invitrogen Bis-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 Invitrogen 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 Invitrogen pre-cut membrane/filter sandwiches, use two thick filter papers.

3) 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

4) Perform the transfer at 15 V (constant) for 15 min if you are using the Bio-Rad Trans-Blot Semi-Dry Transfer Cell. For any other semi-dry transfer cell, follow the manufacturer's recommendations.

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

There are three common explanations:

1) The buffer was accidentally made too dilute, which increases resistance and lowers conductivity and current. Check the transfer buffer and its reagent components, remake, or redilute.

2) The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply. Check the equipment.

3) There is a leak in the blot module. This is indicated by a drastic decrease in current and in buffer volume within the module.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Three possible explanations are:

1) Poor contact between the gel and the membrane: the gel should contact the membrane across the entire surface for good capillary action. To ensure good contact and remove any bubbles, roll a glass pipette over the surface of each layer of the gel/membrane sandwich. Pipet some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the blotting pads need to be fully saturated with transfer buffer (push down with a gloved hand when they are submerged to ensure that there are no air bubbles).

2) Undercompression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with new ones.

3) Overcompression of the gel: A good indication of overcompression is if the gel has been excessively flattened. In the event that the sandwich is overcompressed, remove enough pads so that the blot module can be closed without exerting excess pressure on the gel and membrane. NOTE: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

A number of human alkaline phophatases that could be consuming the WesternBreeze substrate have a molecular weight close to 50 kDa. These would be detected as a band of approximately 54 kDa. These proteins are known to dimerize, and can show as a band of approximately 110 kDa in native gels.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Less-than-optimal transfer in the second gel is not uncommon and frequently requires adjustments in protocol. To achieve similar transfer efficiencies in the two gels you can transfer them one at a time or consider the following suggestions:

Make sure that the blot module is not overfilled with buffer, i.e., fill to point where pads are just covered and no higher. If the buffer level is too high, some current will bypass the gels. If it is overfilled, shorting, arcing, and other problems can occur.

Use antioxidant when transferring reduced proteins.

Increase transfer time by 30 to 60 minutes. The longer run time allows the slower moving proteins time to move out of the gel.

As proteins are being driven out of the gel by the SDS on their surface, it is important that enough is retained to keep them mobile. However, the methanol that is included in the transfer buffer will remove some of the SDS, and the higher the methanol concentration, the more SDS is washed off of the protein. Methanol is included in order to increase binding to nitrocellulose membranes via hydrophobic interactions. However, if using PVDF or nylon membranes, less or even no methanol may be required. 20% methanol is a good starting point for most situations In the event that the transfer is less than ideal, methanol levels should be reduced by at least 50%. The optimal percentage should be determined empirically. Something to keep in mind is that with increased mobility, the proteins may just move straight though the first membrane. In this case, a second membrane may trap these samples.

It is possible to BRIEFLY (1 min) soak the second gel in transfer buffer with 0.01 to 0.02% SDS before assembling the sandwich. This must be a short soak or the proteins will tend to diffuse out of the gel. Adding 0.01-0.02% SDS to the transfer buffer and using it for the transfer will also aid in protein mobility.

Halfway through the transfer process (30 min), swap the front gel and back gel in the blotter. This is fairly easy to do if you just swap pad-paper-gel-membrane-pad sections.

Another recommendation: if you must transfer simultaneously, and want to be able to compare results of both blots, use an internal control in both gels to get an idea of the relative transfer efficiency and to afford some normalization of the results.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

For proteins larger than 100 kDa, 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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane by rolling a glass pipette over the membrane surface.
- Expired or creased membranes used. Use fresh, undamaged membranes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Here are possible causes and solutions:

- Poor contact between the gel and the membrane: The gel should be attached to the membrane through capillary action. To ensure that this happens, make sure that you roll over the surface of each layer of the gel/membrane sandwich with a glass pipette to ensure good contact between the gel and the membrane. It is helpful to use a disposable pipette to place some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the pads need to be fully saturated (push down with gloved hand when they are placed in transfer buffer to make sure there are no air bubbles.)
- Under-compression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with fresh ones.
- Over-compression of the gel: A good indication of over-compression is if the gel has been excessively flattened. In the event that the sandwich is over-compressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and membrane.
Note: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- High ionic strength of the transfer buffer. Prepare the buffer as described in the manual.
- Power supply is operating at a current close to the current limit of the power supply. Use a power supply with higher limits.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Too short a transfer time: Increase the blotting time by 15 minute increments.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Inappropriate amount of SDS: Add 0.01-0.02% SDS to the transfer buffer to facilitate migration of the protein out of the gel.
- Inappropriate methanol content: Decrease the amount of methanol in the transfer buffer.
Note: Higher molecular weight proteins usually do not transfer completely as compared to mid to low molecular weight proteins.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Too long a transfer tim: Shorten the transfer time by 15 minute increments.
- Inappropriate amount of SDS: Do not include any SDS in the transfer buffer.
- Inappropriate methanol content: Add additional methanol to the transfer buffer to increase the binding capacity of the membrane.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Sample overloaded: Decrease the sample load.
- Finally, if using nitrocellulose membrane, switch to PVDF which has a higher binding capacity.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

It is possible that the gel/membrane sandwich was assembled in the reverse direction such that the proteins have migrated out into the buffer. Assemble the blot sandwich in the correct order using instructions provided in the manual.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The most common cause of abnormally high current is the buffer. If the buffer is too concentrated, this leads to increased conductivity and higher current. High current may also occur if Tris-HCl was accidentally substituted for the Tris base required in the transfer buffer. Tris-HCl results in a low buffer pH and leads to increased conductivity and current, and, subsequently, overheating. Check the transfer buffer and its reagent components, re-dilute, or remake the buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- The buffer was accidentally made too dilute, therefore increasing resistance and thus lowering conductivity and current: Check the transfer buffer and its reagent components and then re-dilute it or remake it.
- The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply: Check the equipment.
- There is a leak in the blot module (this is indicated by a drastic decrease in current and in buffer volume within the module): Ensure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer.
- Tape at the bottom of the gel cassette was not removed: Double check that the tape on the bottom of the gel has been removed.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are some suggestions:

- Check the power supply unit.
- Double check that the tape on the bottom of the gel cassette has been removed.
- Make sure that the gel(s) are oriented so that the taller sides of the cassette (with the printing) are facing the outside of the electrophoresis unit.
- Make sure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer. If the wells are not covered, check for leaks and reseal.
- Double check to see if there are any loose electrodes or connections on the Mini Cell unit.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

You may purchase the Invitrogen Power Supply Adapters, Cat. No. ZA10001 to help you connect your leads to the power supply.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

We recommend marking the cassette at the bottom of the wells with a marker pen prior to assembling the Upper buffer chamber. Also, we recommend illuminating the bench area with a light source placed directly behind the XCell SureLock Mini-Cell unit.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Buffers are too dilute. Check buffer recipe; remake if necessary.
- Upper buffer chamber is leaking. Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
- Voltage is set too low. Set correct voltage.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Our Original Bolt Bis-Tris Plus gels (Cat. No. BGxxxxxBOX), discontinued as of December 31, 2014) do not fit in the XCell SureLock Mini-Cell. They need to be run in the Bolt Mini Gel Tank (discontinued as of December 31, 2014). Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX) can be run using the XCell SureLock Mini-Cell (or Mini Gel Tank, Cat. No. A25977).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are the replacement parts we offer for the XCell SureLock Mini-Cell:
Replacement part - Cat. No.
Buffer Dam for XCell SureLock Mini-Cell and XCell II Blot Module - EI0012
L-Buffer Chamber for XCell SureLock Mini-Cell and XCell II Mini-Cell - EI0013
Platinum Wire for XCell SureLock Mini-Cell (12 inch x 2) - EI9022
XCell SureLock Lid - EI0010
XCell SureLock Wedge - EI0011
Buffer Core with Cathode Bar for XCell SureLock Mini-Cell - EI9015
Gel Knife - EI9010
Sponge Pad for Blotting (8) - EI9052

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The XCell SureLock Mini-Cell is impervious to alcohol, but not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene) or acetone.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

You can try the standard recommendation for cleaning which would be washing the unit with a mild detergent and rinsing with deionized water.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

We recommend washing the XCell SureLock Mini-Cell with a mild detergent and rinsing with deionized water after each use.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The XCell SureLock Mini-Cell includes the lid, gel tension wedge, lower buffer chamber, buffer core, buffer dam, and gel knife.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The Precise Tris-HEPES gels are compatible with the XCell SureLock Mini Cell or Mini Gel Tank when used with adaptor plates. Note: Two adaptor plates are required when running just one gel and one adaptor plate is required when running two gels using the XCell SureLock Mini Cell or Mini Gel Tank.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Our Original Bolt Bis-Tris Plus Mini gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) can only be run in the Bolt Mini Gel Tank (discontinued as of December 31, 2014, and will be offered until inventory is depleted).

Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX), as well as our Invitrogen Mini gels and NuPAGE Mini gels can be run using the Mini Gel Tank, or XCell SureLock Mini-Cell. To run these gels using the Bolt Mini Gel Tank (discontinued as of December 31, 2014), upgrading of the tank is necessary by replacing the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732, Cassette Clamp Cam Handle Set). Instructions for replacement of the cam handles can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gel-electrophoresis-chamber-systems/mini-gel-tank/resources-upgrading-bolt-mini-gel-tank.html).

Our Midi gels can be run using the XCell4 SureLock Midi-Cell.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.