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查看更多产品信息 Novex™ Semi-Dry Blotter - FAQs (SD1000)
32 个常见问题解答
以下是可能原因和解决方案：
•膜未完全润湿。一定要按照生厂商的说明预先润湿膜。白点是膜的干燥区域。
•电流过高。应使用具有低电导率的转膜缓冲液，例如使用手册中推荐的缓冲液。
以下是可能原因和解决方案：
•膜在组装到转印三明治中之前变干了。膜在放到三明治中时，应该完全是灰色或呈半透明。如果膜干了，应用甲醇润湿，然后在转膜缓冲液中预平衡。
•不可用乙醇润湿膜。PVDF是疏水性的，需在转印前置于甲醇或乙醇中短暂浸泡。
以下是可能的原因和解决方案：
- 转印过度使蛋白质穿过膜：使用0.2 μm孔径的硝酸纤维素膜代替0.45 μm，或使用结合能力更高的PVDF膜。
- 转膜缓冲液中的甲醇不足：增加甲醇浓度。
- 低分子量蛋白质结合不佳或被洗脱：使用戊二醛将蛋白质交联到膜上，并在洗涤步骤中使用Tween 20。
- 转膜缓冲液含有SDS ：不要在转膜缓冲液中加入SDS。
以下是可能原因和解决方案：
- 空气间隙影响凝胶与膜的接触：将凝胶置于膜上之前用转印滚筒（或干净的试管或吸管）在膜上滚动，然后用转印滚筒或戴有湿手套的手指除去凝胶与膜之间的气泡。若凝胶与膜未接触，则无法转印。
- 电泳条件错误或不理想：电泳条件、样品制备、丙烯酰胺比例和许多其他变量均可影响蛋白质的迁移和分辨率。请查看您的电泳条件。
- 对凝胶的压力不足或过大：遵循使用手册（https://tools.thermofisher.com/content/sfs/manuals/Invitrogen_semidry_blotter_man.pdf）中的挤压指南。压力过小，会使蛋白质在凝胶与膜之间迁移，导致蛋白质条带模糊。压力过大，会使凝胶扭曲。
以下是可能原因和解决方案：
- 电压过低：厚度为1mm的聚丙烯酰胺凝胶（小型和中型凝胶）的转印电压应为20V，E-PAGE为25 V（约15 V/cm场强）。
- 电源不适用于半干转：有些电源在半干转所需的条件下运行时，会自动关闭或熔断保险丝。半干转需要低电压（20 V）和高电流。应与电源生产商联系，确定电源是否适用于半干转。
- 转印时间过短：延长转印时间。标准的转印时间为30-60分钟。
- 转印三明治组装顺序错误：Invitrogen半干转仪的阴极在上、阳极在下。这样才能使蛋白质从凝胶转印到膜上。组装转印三明治时，应仔细按照说明操作。
- 转膜缓冲液的pH与蛋白质的等电点太接近：如果按照使用手册的说明进行配制，则转膜缓冲液应为最佳pH。不要用酸或碱调节pH，因为这会使缓冲液的电导率升高，进而导致转印期间电流过高。
- 转膜缓冲液中的甲醇含量太高： 减少甲醇用量有助于将蛋白质从凝胶中迁出，但会减少蛋白质与硝酸纤维素膜的结合。
- 高比例凝胶可限制转印：较高比例的聚丙烯酰胺凝胶或交联剂可限制蛋白质的洗脱。使用最低比例的丙烯酰胺，有可能将您的蛋白质分离。
- 转印板上有缓冲液，可通过电流，使电流绕过膜组： 每次盖上转印装置盖子前，都要清理下层板。不要过度挤压膜组，否则会将转印滤纸中的转膜缓冲液挤出，形成水流并使电流通过。
以下是经过优化的Bio-Rad半干转设备转印方案。Tris-甘氨酸凝胶转印可使用NuPAGE转膜缓冲液。
1.工作转膜缓冲液：含10%甲醇、1:1,000抗氧化剂的2X NuPAGE转膜缓冲液（Bis-Tris 50 mM和Bicine 50 mM）。如果您需要使用NuPAGE 20X转膜缓冲液（货号NP0006）配制100 mL工作缓冲液，则按下述配方混合：10 mL 20X转膜缓冲液、10 mL甲醇、100 µL抗氧化剂和80 mL去离子水。
2.滤纸：三明治中经转膜缓冲液浸泡的滤纸，是半干转槽中唯一的缓冲液储层。若使用Invitrogen预切膜/滤纸三明治，则凝胶（或膜）两侧至少需要使用3层滤纸（每张滤纸厚0.4 mm）。在组装一个凝胶/膜三明治时，在工作转膜缓冲液（按步骤1配制）中预先浸泡6张Invitrogen滤纸（或2张厚滤纸）和1张膜，然后按以下顺序在阳极板上组装三明治：滤纸--滤纸--滤纸--膜--凝胶--滤纸--滤纸--滤纸。
3.转印条件：我们发现在Bio-Rad半干转仪中使用NuPAGE转膜缓冲液进行转印的最佳条件是电压15 V、转印时间15-30分钟。其他生产商生产的半干转设备，应按照其相应的说明进行使用。
4.转印大分子量蛋白质（≥100 kDa）时，可在组装三明治前，将凝胶置于含0.02-0.04% SDS 的2X NuPAGE转膜缓冲液（无甲醇）中预平衡10分钟。请注意，转印Tris-甘氨酸凝胶时，使用Bio-Rad Trans-Blot SD半干转槽搭配NuPAGE转膜缓冲液的转印效率低于使用XCell II转印模块（半湿转）搭配Tris-甘氨酸转膜缓冲液（货号LC3675）。
对于E-PAGE 48凝胶的半干转，我们建议使用含15%甲醇的2X NuPAGE转膜缓冲液；对于E-PAGE 96凝胶的半干转，我们建议使用无甲醇的2X NuPAGE转膜缓冲液。在25V恒定电压条件下转印30-60分钟。请参考E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第33页。
E-PAGE凝胶转印的推荐方法是使用iBlot/iBlot 2干转系统进行干转（见E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第27页）。请注意，该系统仅适用于E-PAGE 48凝胶。也可采用Invitrogen半干转仪（货号SD1000，第33页）进行半干转或采用半湿转法（第37页）。
可以，我们建议使用含10%甲醇的2XTris-甘氨酸转膜缓冲液，在20V恒定电压下转印30-60分钟。
我们建议使用2X NuPAGE转膜缓冲液，在20V恒定电压下转印30-60分钟。
NuPAGE Bis-Tris凝胶在Invitrogen半干转仪中的转印效率不如在XCell II转印模块中高。如果您决定使用Invitrogen半干转仪进行NuPAGE -Tris凝胶转印，则应使用下述实验方案以确保蛋白质的有效转印。
1.如下所述，使用20X NuPAGE转膜缓冲液制备100 mL的2X NuPAGE转膜缓冲液：
NuPAGE转膜缓冲液（20X）10.0 mL
NuPAGE抗氧化剂（用于还原型样品）0.1 mL
甲醇10.0 mL
去离子水79.9 mL
总体积100 mL
如果您转印的是大分子蛋白质，请参见下方备注。
2.在转膜缓冲液中浸泡滤纸和印迹膜。如果您在使用Invitrogen预切膜/滤纸三明治，则应在凝胶或膜的外侧分别使用3张滤纸（每张滤纸厚0.4 mm）。如果您未使用Invitrogen预切膜/滤纸三明治，则使用2张厚滤纸。
3.将凝胶置于转膜缓冲液（中型凝胶使用100 mL，小型凝胶使用50 mL）中，放置在定轨摇床上平衡10分钟，从而去除转印液中的盐，以避免电导率和产热的增加。
4.如下所述，在阳极板上装配凝胶/膜/滤纸三明治：
滤纸
滤纸
滤纸
滤纸
膜
凝胶
凝胶
凝胶
5.在20 V恒定电压条件下转印30-60分钟。
备注：转印大分子量蛋白质（>100 kDa）时，可在装配三明治前，将凝胶置于含0.02-0.04% SDS 的2X NuPAGE转膜缓冲液（无甲醇）中预平衡10分钟。
在半干转免疫印迹分析中，转膜缓冲液的浓度必须是湿转的2倍（即，2X），从而确保较小的缓冲液体积中含有足够的缓冲离子。
Invitrogen半干转仪可同时转印1-2块中型凝胶、1-4块小型凝胶或1-2块E-PAGE凝胶。
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.