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View additional product information for Novex™ Semi-Dry Blotter - FAQs (SD1000)
18 product FAQs found
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.
If using Invitrogen Semi-Dry Blotter (Cat. No. SD1000), 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 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 kD 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).
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.
No, most transfer applications require high current and therefore, we recommend using a high current power supply like PowerEase Touch 350W Power Supply.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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 (http://tools.thermofisher.com/content/sfs/manuals/Invitrogen_semidry_blotter_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Over-transfer through the membrane: Use 0.2 µm pore size nitrocellulose instead of 0.45 µm, or use PVDF with a higher binding capacity.
- Not enough methanol in the transfer buffer: Increase the concentration of methanol.
- Low molecular weight proteins are not binding well or are being washed away: Use glutaraldehyde to crosslink the proteins to the membrane and use Tween 20 in the wash steps.
- SDS is included in the transfer buffer: Do not use SDS in the transfer buffer.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Air spaces are interfering with contact between the gel and the membrane: Roll the membrane with a blotting roller (or a clean test tube or pipet) before placing the gel on the membrane, and then remove any air bubbles between the gel and membrane with a blotting roller or a wet gloved finger. Transfer will not occur where the gel is not in contact with the membrane.
- Electrophoretic conditions were incorrect or not ideal: Running conditions, sample preparation, percentage acrylamide, and many other variables can affect the migration and resolution of proteins. Please review your electrophoresis conditions.
- Under- or over-compression of gel: Follow the compression guidelines in the manual (http://tools.thermofisher.com/content/sfs/manuals/Invitrogen_semidry_blotter_man.pdf). Too little compression can allow proteins to migrate between the gel and membrane, causing protein band smearing. Too much compression can distort the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Voltage is too low: 1 mm thick polyacrylamide gels (Mini and Midi Gels) should be transferred at 20 V, E-PAGE Gels at 25 V (approximately 15 V/cm field strength).
- Power supply is inappropriate for semidry transfer: Some power supplies will shut off or blow a fuse when run at the conditions required for semi-dry transfer. Semi-dry transfer requires low voltage (20 V) and high current. Check with the manufacturer of the power supply to determine whether it is appropriate for semi-dry transfer.
- Transfer was performed for too short a time: Increase the amount of time for transfer. Typical transfer times range from 30 to 60 minutes.
- Transfer sandwich was assembled in the wrong order: The Invitrogen Semi-dry Blotter is configured with the cathode on the top, and anode on the bottom. This results in a downward transfer of proteins from the gel onto the membrane. Follow the instructions carefully when assembling the transfer sandwich.
- The pH of the transfer buffer is too close to the isoelectric point of the protein: The transfer buffers should be at the optimal pH if prepared as described in this manual. Do not adjust the pH with acid or base as this will increase the conductivity of the buffer and result in higher current during transfer.
- Too much methanol is present in the transfer buffer: Reducing the amount of methanol can help elute proteins from the gel, but can reduce binding to nitrocellulose membranes.
- High-percentage gels restrict transfer: Higher percentage acrylamide or crosslinkers can restrict elution of proteins. Use the lowest percentage acrylamide possible to separate your proteins.
- Puddles of buffer were present on the plates, allowing the current to bypass the stack: Always clean up the lower plate before closing the lid of the transfer apparatus. Do not squeeze the stack excessively, as this removes transfer buffer from the blotting paper and also creates puddles that the current can pass through.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
- 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.
- 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
- 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.
- 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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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 (http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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 http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf). Please note that this works only for E-PAGE 48 gels. Semi-dry blotting using the Invitrogen Semi-Dry Blotter, Cat. No. SD1000 (Page 33), or semi-wet blotting (Page 37) may also be performed.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, we recommend using 2X Tris-Glycine Transfer Buffer containing 10% methanol and performing the transfer at 20 V (constant) for 30-60 minutes.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend using 2X NuPAGE Transfer Buffer and performing the transfer at 20 V (constant) for 30-60 minutes.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
NuPAGE Bis-Tris Gels do not transfer efficiently using the Invitrogen Semi-Dry Blotter as compared to blotting with XCell II Blot Module. If you decide to use the Invitrogen Semi-Dry Blotter for NuPAGE Bis-Tris Gels, use the protocol provided below to ensure efficient transfer of proteins:
- 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.
- 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.
- 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.
- 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
- 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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The Invitrogen Semi-Dry Blotter allows simultaneous transfer of 1-2 Midi Gels, 1-4 Mini Gels, or 1-2 E-PAGE gels.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Do not use ethanol or other organic solvents to clean the Invitrogen 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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.