Search
Search
查看更多产品信息 BenchPro™ 4100 Western Card - FAQs (WP1001)
67 个常见问题解答
是的，印迹膜将被保留在程序中最后使用的试剂里，无论最后是什么试剂。为了将印迹膜保留在洗涤缓冲液中，只需以洗涤代替润洗作为最后一步。
以下建议可能会有所帮助：
•使用含PBS的封闭剂，不要使用Tween 20或BSA。
•应使用含0.01% Tween 20的PBS稀释一抗和二抗，现用现配。
•最后2次洗膜时，仅使用PBS以去除Tween 20，从而避免产生背景。
我们建议断开电源并重启仪器。
这可能是由于以下两个原因：
•交流电电源线未连接或使用了错误的电源线。
•保险丝熔断。
以下是可能原因和解决方案：
- Western卡片没有在正确的位置： 将卡片拉出并重新插入，确保将卡片一直插到底，直到听到“咔哒”声。
- 溶液吸头堵塞： 检查溶液，特别是所有牛奶封闭液，查看是否存在可堵塞吸头的大颗粒。
- 卡片有缺损： 尝试使用新卡片或尽可能不同批号的卡片[尝试使用新卡片，如果条件允许，尽可能使用不同批号的卡片。]。如果您怀疑卡片有问题，请发送邮件至techsupport@lifetech.com联系技术支持。
- 检查仪器的压力值和负压值： 如果仪器的压力值明显低于248 kPa且负压值明显低于48 kPa，则可能存在硬件问题。请发送邮件至techsupport@lifetech.com联系技术支持。
以下是可能原因和解决方案：
- SDS残留或转印后蛋白质与膜结合较弱： 遵循免疫检测前的膜准备说明。
- 封闭时间过短或洗膜时间过长： 应确保每一步都达到指定时间。
- 一抗对蛋白标准品具有亲和力： 向蛋白标准品生产商咨询蛋白标准品与一抗的同源性。
- 膜被指纹或角蛋白污染： 始终佩戴干净的手套并使用镊子来处理膜。处理膜时，仅触碰膜的边缘。
以下是可能原因和解决方案：
- 膜过度曝光或在曝光过程中变湿： 缩短曝光时间或使信号进一步衰减。将膜放在透明薄膜中防止泄漏，并在曝光前从边缘吸去过量的底物。
- 封闭或洗膜时间太短： 应确保每一步都达到指定时间。
- 一抗和/或二抗浓度过高： 通过点印迹法确定最佳抗体浓度，必要时可稀释抗体。
- 接触膜的溶液、托盘或管子污染： 使用干净的玻璃器皿和纯水制备溶液。使用纯水彻底清洗托盘。尽管使用温和去污剂清洗并用去离子水冲洗过的所有试剂瓶和试管都可以重复使用，但是，为防止任何试剂发生交叉污染，我们不建议重复使用25 ml试管。始终佩戴干净的手套。用镊子处理膜。
- 蛋白质上样量过高： 减少上样量或稀释样品浓度。
以下是可能原因和解决方案：
- 检测步骤缺失或检测试剂无效： 仪器实验方案中不包含检测步骤。仪器实验方案完成后，应使用标准检测试剂和方案进行手动检测。应确认检测试剂是有效的。
- 在检测试剂中孵育不充分： 在达到可接受的信噪比时，从检测试剂中取出印迹膜。
- 试剂加入顺序错误： 为确保每一步都正确，应按照使用手册第13页所述的顺序加入试剂。
- 转印效果差或转印不完全： 应确保转印装置和膜三明治组装正确。使用合适的转印时间。转印后，对膜进行染色以衡量转印效率。
- 目标蛋白跑出凝胶： 使用阳性对照和/或分子量标记物来匹配凝胶分离范围和转印蛋白的大小。转印后，对膜进行染色以衡量转印效率。
- 样品太稀： 增加蛋白质样品在凝胶上的上样量。
- 蛋白质保留较差或蛋白质与膜的结合较弱： 转膜液应含10–20%甲醇。使用具有适当结合能力的膜。
- 一抗或二抗失活或稀释过度： 通过点印迹法确定抗体活性。必要时可增加抗体浓度。
以下是可能原因和解决方案：
•未取下瓶盖。应确认将试剂瓶和试管放入试剂托盘时，已取下盖子。
•卡片方向错误。将卡片插入到BenchPro 4100卡片处理站中，使吸头插入试管托盘上的试剂瓶和试管中。使用卡片对齐指南，确保将卡片正确插入槽内。当卡片正确插入槽内时，您将听到“咔哒”声。
我们建议您在开始实验方案之前，确认已按正确顺序加入了卡片、试剂和试剂瓶。如果在运行开始后中止运行，将需要重新开始。如果在运行开始后几分钟内中止运行，请不要重复使用卡片。
请参考使用手册(https://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf)第30页的错误信息列表及相应措施。
以下是可能原因和解决方案：
•交流电电源线未连接。检查交流电电源线两端的连接情况。使用正确的电源线。
•保险丝熔断。更换保险丝（见使用手册(https://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf)第31页）。如果在确认使用了正确电源线并更换保险丝后，问题仍然存在，请发送邮件至techsupport@lifetech.com联系技术支持。
从功能上来说，母步骤和子步骤的执行是相同的。但是，您需要在编辑一个母步骤后才能编辑子步骤（否则会显示错误信息）。如果您选择“母步骤”，然后立即选择“子步骤”，则“母步骤”会默认为1分钟，但不会保存“时间”，保存的方案将显示为进入“母步骤”。当浏览保存后的实验方案时，会看到上述情况。
BenchPro 4100可通过将一抗孵育步骤的时间延长至几百分钟来实现过夜孵育。其他试剂应从一开始就要装载到仪器中，因为无法在第二天停止运行、取出卡片装入新试剂，然后使用前一天使用的卡片继续执行相同方案。如果担心试剂在室温中过夜会出现问题，可在冷室或Deli冷却器中运行仪器。
很遗憾，系统中止后，不会将试剂送回至原试管中；它们将随机排出卡片。
运行后，每种缓冲液约有0.5–1 mL将保留在卡片和吸管中。
快速程序的检测灵敏度通常可达到标准WesternBreeze免疫检测方案的70–80%。
保险丝为250 V、1.25 A。我们不提供保险丝独立产品，但可从电子产品供应商处购买，如RadioShack。
不需要，但是请确保将试剂瓶和试管装载到与将被使用的卡片位于同一排的托盘中。设备可自动检测到哪个插槽含卡片，只有含卡片的插槽在运行期间是可发挥功能的。
我们尚无任何经过验证的方案，但是我们建议用箔纸覆盖Western卡片或尽可能在暗室中运行实验。
可以，处理器经过编程，可将使用过的试剂返还到原试管中。
操作温度范围为4-40℃。所以，可根据需要，在冷室或保温箱中操作仪器。但是，我们在冷室中只测试过几天。
不能，该卡片不可重复使用。仪器能够检测出使用过的卡片，卡片右下角的颜色指示点会在卡片被使用后变成红色。
无需同时使用4张卡片。您可使用1张卡片或同时使用2-4张卡片。
8.5 cm x 8.5 cm是可处理的最大膜尺寸。
可以，因为每张卡片分开处理。
该仪器可处理1-4张Western卡片。每张卡片可同时处理1或2张膜，背对背放置。因此，该系统每次运行可同时处理最多8张膜。
BenchPro 4100卡片处理站采用3种最常用的免疫印迹分析方案进行预先编程：2种用于Western Breeze免疫检测，1种用于标准ECL检测。这些方案经过编程，可将一抗送还到原试管中。此外，超过17个自定义方案可被保存在系统记忆中。对自定义方案进行编程，可根据需要将任何试剂送还到原容器中。
我们不提供自动排水用6 ft排水管。它的规格为¼” OD x 1/8” ID，您可登录网站http://www.mcmaster.com/#catalog/119/136/=q5ikda进行购买。
25 mL试管可单独购买，规格为50管/包（货号WP3001）。但是，我们不提供试剂瓶的独立产品。您可从Cole Parmer公司购买。
250 mL试剂瓶（套装1）（货号EW-06019-76） (http://www.coleparmer.com/Product/Bottles_Squre_HDPE_250ml_10_pk/EW-06019-76
125 mL试剂瓶（套装2）（货号EW-06254-20）(http://www.coleparmer.com/Product/Thermo_Scientific_Nalgene_Sterile_PETG_Media_Bottles_125_mL_48_box/EW-06254-20)
用湿布清理BenchPro 4100卡片处理站的表面。不要使用刺激性去污剂或溶剂清洁设备。每次使用后，用水冲洗试剂托盘组件和废水池。如果瓶子和管子是可重复使用的，则用温和去污剂清洗，并用超纯水彻底冲洗。不要将托盘或试剂瓶和试管进行高压灭菌。尽管所有试剂瓶和试管在使用温和去污剂清洗并用去离子水冲洗后均能重复使用，但是我们不推荐重复使用25 mL试管，以防止任何试剂发生交叉污染。
是的，BenchPro 4100卡片处理站于2014年12月31日起停产。之前购买的所有仪器将享受生产商的一年保修。为支持已购买仪器的客户，在2017年12月31前，我们继续生产和销售BenchPro 4100的耗材。
可以，蛋白质免疫印迹处理设备适用于Alexa Fluor 680和790标记的二抗，其灵敏性与手动处理相近或比手动处理更好。参见该链接(https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/ibind-western-system.html?icid=fr-western-3)中的图2。
Yes, the blot will be held in the last reagent programmed, regardless which reagent that is. For the blot to be left in wash buffer, just make wash the last step instead of rinse.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are some recommendations that may help:
- Use a PBS-based Blocking reagent and do not use Tween 20 or BSA.
- Make sure that the Primary and Secondary antibodies are freshly diluted in 0.01% Tween 20 in PBS
- For the last 2 washes, use only PBS to get rid of the Tween 20, thus avoiding background.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We would recommend shutting off the power and restarting the instrument.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
This could be due to the following reasons:
- AC power cord is not connected or incorrect cord was used.
- Fuse has blown.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- The Western Card may not be in the right position: Pull it out and insert it again to make sure it is inserted all the way down until you hear a click sound.
- Solution intake tips could be clogged: Check your solutions, especially any milk blocking buffers, to see if there are large particles present that could be blocking the tips.
- Defective cards: Try a new card, or a card from a box with a different lot number if possible. If you suspect a problem with your cards, please contact Technical Support at techsupport@thermofisher.com.
- Check instrument pressure and vacuum value: If numbers are significantly below 248 kPa for instrument pressure and below 48 kPa for vacuum, there could be a possible hardware issue. Please contact Technical Support at techsupport@thermofisher.com .
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Membrane is contaminated by fingerprints or keratin proteins_ Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Film overexposed or became wet during exposure: Decrease exposure time or allow signal to further decay. Prevent leakage of solutions by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
- Short blocking time or washing time: Perform each step for the specified amount of time.
- High concentration of primary and/or secondary antibody: Determine optimal antibody concentration by performing a dot blot. Decrease antibody concentration as necessary.
- Membrane, solutions, trays, or vials are contaminated: Use clean glassware and purified water to prepare solutions. Rinse the tray thoroughly with purified water. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 ml vials to prevent any reagent cross-contamination. Wear clean gloves at all times. Use forceps when handling membranes.
- Protein is overloaded: Reduce load or dilute the sample.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Detection step missed or detection reagents not working: The detection step is not included in the instrument protocol. After the instrument protocol is complete, perform the detection step using your standard detection reagents and protocol manually. Make sure the detection reagents are functional.
- Insufficient incubation with detection reagent: Remove the blot from the detection reagent when signal-to-noise ratio is acceptable.
- Incorrect order of reagents added: Be sure to add the reagents in the order described on page 13 of the manual (https://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf) to ensure all steps are performed correctly.
- Poor or incomplete transfer: Make sure transfer apparatus and membrane sandwiches are assembled correctly. Use appropriate transfer times. After blotting, stain membrane to measure transfer efficiency.
- Protein of interest ran off the gel: Use positive control and/or molecular weight marker to match gel separation range to size of protein being blotted. After blotting, stain membrane to measure transfer efficiency.
- Sample was too dilute: Load a larger amount of protein onto the gel.
- Poor retention of proteins or protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol. Use membranes with appropriate binding capacity.
- Inactive or overly dilute primary or secondary antibody: Determine antibody activity by performing a dot blot. Increase antibody concentration as necessary.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Bottles have caps: Make sure that the reagent bottles and vials are uncapped when placed in the reagent tray.
- The card is not oriented correctly: Insert the card into a slot in the BenchPro 4100 Card Processing Station such that the tips are inserted into the reagent bottles and vials placed in the reagent tray. Use the Card Alignment Guide to ensure the card is placed correctly in the slot. When the card is inserted correctly in the slot, you will hear a hard click.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend making sure that you have added the card, reagents, and reagent bottles in the correct order prior to starting the protocol. If the run is aborted after the run has begun, you need to restart the run. Do not reuse the card, if the run is aborted within a few minutes of starting the run.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Please refer to the list of error messages and actions to take on page 30 in the manual (http://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- AC power cord is not connected. Check AC power cord connections at both ends. Use the correct cords.
- Fuse has blown. Replace the fuse (see page 31 in the manual http://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf). If the problem still persists after verifying that correct power cord is used and the fuse is replaced, contact Technical Support at techsupport@thermofisher.com.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Functionally, steps and sub-steps will perform the same. However, you cannot program a sub-step without first programming a step (an error message will display if you try). If you select 'step' and then immediately select 'sub-step', the 'step' will default to 1 min for the 'minutes' but no 'times' will be saved and the protocol that is saved will display the entry as a 'step'. This can be seen when the protocol is viewed again after it is saved.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The BenchPro 4100 allows for overnight incubation by increasing the time of the primary antibody incubation step to several hundred mins. The rest of the reagents need to be loaded in the instrument from the beginning, though, as there is no way to halt the run the next day, remove the card to load new reagents, then continue the same protocol with the same card as the previous day. If there is concern about reagents sitting overnight at room temperature, the instrument can be run in a cold room or deli cooler.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Unfortunately, the abort does not send the reagents back to where they came from; they randomly drain through the card.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
About 0.5-1 mL of each buffer will remain in the card and tips after the run.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The faster program will generally give sensitivity in detection that is 70-80% of the standard WesternBreeze immunodetection protocol.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The fuse is 250 V, 1.25 A. We do not offer it as a standalone product, but it can be purchased from electronics vendors such as RadioShack.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
No, but please ensure that the reagent bottles and vials have been loaded in the same tray row as the cards that will be used. The unit automatically detects which slots contain cards and only the slots containing cards are functional during processing.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We do not have any validated protocols for this, but we suggest covering the Western Card with foil or running the experiment in a dark room if possible.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, the processor can be programmed to return reagents back into the same vials after use.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The operating temperature range is 4-40 degrees C. So the instrument can be operated in the cold room or incubator if needed. However, we have only tested for a few days in the cold room.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
No, the cards are not meant to be reused. The instrument detects used cards by a color indicator spot at the lower right hand corner of the card that turns red after the card is used.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
There is no need to use all 4 cards at a time. You can use one card or 2-4 cards at a time.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
8.5 cm x 8.5 cm is the maximum membrane size that can be processed.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, as each card is filled from a separate set of vials.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The instrument has the capacity to process 1-4 Western Cards. Each card could process 1 or 2 membranes at a time, placed back to back. Therefore, the system has the capacity to simultaneously process up to 8 membranes in each run.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The BenchPro 4100 Card Processing Station is pre-programmed with three of the most popular western protocols: two for Western Breeze immunodetection and one for standard ECL detection. These protocols are programmed to return the primary antibody to the original vial. Additionally, 17 more custom protocols can be stored in the system's memory. The custom protocols can be programmed to return any of the reagents back to the original container, if desired.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We do not offer the 6 ft Drain tubing for Auto Drain. It is 1/4 inch OD x 1/8 inch ID and you may purchase it from http://www.mcmaster.com/#catalog/119/136/=q5ikda.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The 25 mL Reagent Vials can be purchased separately as a set of 50 vials/package (Cat. No. WP3001). However, we do not offer the Reagent Bottles as standalone products. You may purchase them from Cole Parmer:
- 250 mL Reagent Bottles (Set 1) (Cat. No. EW-06019-76) (https://www.coleparmer.com/i/cole-parmer-graduated-square-hdpe-wide-mouth-bottle-250-ml-10-pk/0601976?searchterm=EW-06019-76)
- 125 mL Reagent Bottles (Set 2) (Cat. No. EW-06254-20) (https://www.coleparmer.com/i/thermo-scientific-nalgene-2019-0125-sterile-petg-media-bottles-125-ml-48-box/0625420?searchterm=EW-06254-20)
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Clean the surface of the BenchPro 4100 Card Processing Station with a damp cloth. Do not use harsh detergents or solvents to clean the unit. Rinse the reagent tray assembly and waste basin with water after each use. If bottles and vials are to be reused, clean with mild detergent and rinse thoroughly with ultrapure water. Do not autoclave trays or reagent bottles and vials. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 mL vials to prevent any reagent cross-contamination.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, the BenchPro 4100 Card Processing Station has been discontinued as of December 31, 2014. The current BenchPro 4100 Western Cards (https://www.thermofisher.com/order/catalog/product/WP1001) and Reagent Vials (https://www.thermofisher.com/order/catalog/product/WP3001) will continue to be available for purchase. We offer the new iBind Western Device (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-detection/ibind-western-system.html) as an alternative system for automated western blot processing.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, western blot processing instruments work well with Alexa Fluor 680 and 790 labeled secondary antibodies and give similar or better sensitivity compared to manual processing. See Figure 2 in the following link (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/ibind-western-system.html?icid=fr-western-3).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, western blot processing instruments work well with WesternDot reagents.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.