XCell SureLock™ Mini-Cell und XCell II™ Blot Modul

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.