Western Blotting Support—Troubleshooting

• Increase voltage, current or length of time for transfer
• SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2x Transfer buffer (without methanol) containing 0.02–0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01% SDS.
• Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10–20% and that high-quality, analytical grade methanol is used.

• Decrease voltage, current or length of transfer time
• Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10–20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
• Make sure that the SDS concentration (if added) in the transfer buffer is proper, don’t use more than 0.02–0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
• Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 μm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 μm pore size membrane.

The fading is most likely due to detergent in the western blocking/washing solutions that can remove some of the proteins from the membrane. The dye itself will not wash off of the proteins because it is covalently bound. We have found that smaller pore size membranes retain the proteins better during blocking and wash procedures, and hence recommend use of 0.2 µm instead of 0.45 µm membranes for best resolution and protein retention. After transfer, it is a good idea to circle the pre-stained bands with a pencil on the membrane, so band positions can be identified after blocking and processing.

Here are possible causes and solutions:

Here are possible causes and solutions:

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.

We recommend discarding the buffer and remaking it after rechecking the reagents and the water purity. We do not recommend adjusting the pH with acid or base, as this will increase the conductivity of the buffer and result in higher current during the transfer.

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.

Here are possible causes and solutions:

Here are possible causes and solutions:

Note: Higher molecular weight proteins usually do not transfer completely as compared to mid to low molecular weight proteins.

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.

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:

Note: The height of the uncompressed pads should be 0.5–1.0 cm above the level of the sealing gasket.

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.

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.

• Increase the Tris-Glycine transfer buffer pH to 9.2, allowing all the proteins below 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. 

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.

Here are possible causes and solutions:

Here are possible causes and solutions:

Here are possible causes and solutions:

• Membrane was dried out before it was added to the transfer sandwich. Membrane should be completely gray and slightly translucent when added to the sandwich. If it has dried out, rewet in methanol and equilibrate in transfer buffer.
• Alcohol was not used to prewet the membrane. PVDF is hydrophobic and requires a short soak in methanol or ethanol prior to transfer.

Here are possible causes and solutions:

• Membrane was not thoroughly wetted. Always pre-wet the membrane according to the manufacturer’s instructions. White spots indicate dry areas of the membrane.
• Too much current. Use a low conductivity transfer buffer such as those recommended in this manual.

This indicates an incomplete electric circuit and here are possible causes and solutions:

This indicates an open electrical circuit during the run. It occurs if the lid opened during the run.

Close the lid and continue the run by briefly pressing the Start/Stop Button or restart the run by pressing and holding the Start/Stop Button.

This is most likely due to incorrect placement of the top Stack. Be sure the iBlot™ Cathode Stack, Top is placed correctly with the copper electrode side facing up. Avoid placing the top stack in the inverted position.

Longer transfer times result in the deposition of copper ions, causing the blue discoloration. Be sure to perform the transfer for the recommended time for each gel type.

The green discoloration is due to copper ions from the iBlot™ Transfer Stacks being carried by liquids and deposited on the membrane. These deposits do not interfere with downstream processes. The stained regions can be cut away, but membrane washing typically results in their removal. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. 

This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the iBlot™ Cathode, Top and Anode Bottom stacks. Always maintain the membrane size identical to the transfer stack. Transfer quality is not affected by smaller gel size compared to the membrane.

This could be due to no current passing through or because an incorrect voltage Method was used. Make sure that the electrical circuit is complete and current is flowing through the device. Please check to make sure that the correct voltage Method is used (see page 13 in the manual). 

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 using the De-bubbling Roller for E-PAGE™ gels or Blotting Roller for other gels.
• Expired or creased membranes used. Use the iBlot™ Gel Transfer Stacks before the expiration date printed on the package. 

This could happen if an incorrect voltage Method was used or if inappropriate transfer conditions were used. Make sure that the voltage Method and run time used is correct, based on the gel type, as described on page 13 in the manual.

For mini or midi gels:

• Perform an ethanol equilibration step as described on page 28 in the manual to improve transfer.
• Use a lower gel percentage to separate the high–molecular weight proteins.
• Increase the transfer time in 30-second increments.

For E-PAGE™ gels:

• Increase the transfer time in 30-second increments.
• Use Method P2 for 8 minutes. 

Note: It is normal for some proteins to remain in the gel because some high–molecular weight proteins do not transfer completely using the iBlot™ Gel Transfer Device, compared to semi-wet transfer apparatus. 

This could happen if the transfer time used is too long. We recommend reducing the transfer time by 30 second increments.

Note: Pre-stained markers are charged, so tend to blow-through more than regular proteins. 

This indicates that a non-uniform electric field was created around the wells. Ensure that the well protrusions on the E-PAGE™ gel are properly flattened using the De-bubbling Roller. To ensure the best blotting results, we recommend using the De-bubbling Roller with E-PAGE™ gels. If you use the Blotting Roller with E-PAGE™ gels, be sure to follow the recommendations on page 22 of the manual to obtain good results. 

This is likely due to the PVDF membrane being dry or partially dry. Regions where PVDF membranes are dry appear whiter than places where the membrane is wet. Remove the membrane and reactivate in 100% methanol, and rinse in water before reapplying to the transfer stack. 

This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze™ wash solutions. 

This is most likely due to the protein load being too high, as a result of which detection is not within the linear range. Since the immunodetection sensitivity is higher for dry blotting with the iBlot™ Gel Transfer Device than for semi-dry or wet blotting, we recommend that you decrease the protein load, use more diluted antibody, or perform detection for shorter time. You may need to perform some optimization based on your initial results. 

It is possible that the PVDF membrane got displaced during handling or shipment. The activated PVDF membrane is transparent, making it difficult to see. If it is not present on top of the stack, we recommend examining the aluminum seal of the Anode Stack (clear tray) to make sure that the PVDF membrane has not adhered to the seal. If the membrane has adhered to the seal, we suggest reactivating the membrane with pure methanol, then rinsing it well in distilled water, and carefully placing it on the stack. The performance will not be affected by the reactivation process.

The iBlot™ device must be connected directly to the computer for the firmware upgrade to work. Do not use a USB hub.

In order to make a loop-back test you first need to make sure that the drivers and COM port have been successfully installed.

1. With the USB Serial drivers installed, connect your iBlot™ device to your computer’s USB port.
2. Check in Windows Device Manager (Start -> Control Panel -> System -> Hardware tab -> Device Manager) under Ports (COM & LTP) to see if the USB Serial Port (COM) has been successfully created. If the COM port is not listed in the Device Manager, it has not been successfully created. You can install the driver manually from http://www.ftdichip.com/Drivers/VCP.htm
3. If multiple devices are connected to one USB serial port, you may need to disconnect other devices.

If the above steps don’t help, another simple option that may solve the issue would be to try a different USB cable. 

This could happen due to the USB cable disconnecting during the updating process. Please try again with another computer and a different USB cable.

This is most likely due to uneven current distribution during transfer, possible caused by bad electrodes. We recommend getting new electrodes (Cat. No. IB1002).

This indicates an incomplete electric circuit. Here are possible causes and solutions:

This could be caused by incorrect placement of the Top Stack. Please check to make sure that the Top Stack is placed correctly with the copper electrode facing up. Avoid placing the top stack in the inverted position. 

Longer transfer times result in the deposition of copper ions. Be sure to perform the transfer for the recommended time for each gel type. 

The green discoloration is due to copper ions carried with liquids that get deposited onto the membrane. These deposits do not interfere with downstream processes. The stained regions can be cut away, but membrane washing typically results in their removal. 

This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the Top and Bottom stacks. This can be avoided by maintaining the membrane size identical to the transfer stack. Transfer quality is not affected by smaller gel size compared to the membrane. 

This could be due to no current passing through or because an incorrect voltage Method was used. Make sure that the electrical circuit is complete and current is flowing through the device. Please check to make sure that the correct voltage Method is used (see page 17 in the manual). 

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 using the Blotting Roller.
• Expired or creased membranes used. Use the iBlot™ 2 Transfer Stacks before the expiration date printed on the package. 

This could happen if an incorrect voltage Method was used or if inappropriate transfer conditions were used. Make sure that the voltage Method and run time used is correct, based on the gel type, as described on page 17 in the manual. 

For mini or midi gels:

• Perform an ethanol equilibration step as described on page 35 in the manual to improve transfer.
• Use a lower gel percentage to separate the high–molecular weight proteins.
• Increase the transfer time in 30-second increments.

For E-PAGE™ gels:

• Increase the transfer time in 30-second increments.
• Use Method P3 for 8 minutes.

Note: It is normal for some proteins to remain in the gel because some high–molecular weight proteins do not transfer completely using the iBlot™ 2 Gel Transfer Device, compared to semi-wet transfer apparatus. 

This could happen if the transfer time used is too long. We recommend reducing the transfer time by 30 second increments.

Note: Pre-stained markers are charged, so tend to blow-through more than regular proteins. 

This indicates that a non-uniform electric field was created around the wells. Ensure that the gel is properly flattened by using the Blotting Roller. Follow the recommendations on page 19 in the manual to obtain good results. 

This is likely due to the PVDF membrane being dry or partially dry. Regions where PVDF membranes are dry appear whiter than places where the membrane is wet. Remove the membrane and reactivate in 100% methanol, and rinse in water before reapplying to the transfer stack. 

This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze™ wash solutions. 

This is most likely due to the protein load being too high, as a result of which detection is not within the linear range. Since the immunodetection sensitivity is higher for dry blotting with the iBlot™ 2 Gel Transfer Device than for semi-dry or wet blotting, we recommend that you decrease the protein load, use more diluted antibody, or perform detection for shorter time. You may need to perform some optimization based on your initial results. 

Here are possible causes and solutions:

This is most likely due to inefficient binding of some low-molecular weight proteins (<25 kDa) to PVDF membrane. We recommend combining ethanol and Thermo Scientific™ Pierce™ 1-Step Transfer Buffer in a 15:85 ratio before equilibrating filter paper and membrane (e.g., 15 mL of ethanol with 85 mL of Thermo Scientific™ Pierce™ 1-Step Transfer Buffer).

PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking agent 2–5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, reducing non-specific interactions with the primary antibody. Examples of blocking agents are nonfat dry milk, BSA, and casein.

The most probable cause of the swirled artifact is the way the membrane is moved around in the staining solution. Using an orbital shaker can cause this effect. To eliminate these background patterns, we recommend switching to a rocker type shaker or combination of rocker and reciprocal motions to ensure even sloshing of the staining solution over the membrane.