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查看更多产品信息 Novex™ AP Mouse Chemiluminescent Detection Kit - FAQs (SLF1021)
12 个常见问题解答
以下是可能原因和解决方案：
- 转印效果差或转印不完全： 重复转印。
- 膜的转印垫不干净或污染： 使用转印垫前，用去污剂浸泡，然后用纯水彻底清洗。转印垫破损或变色后，则更换新的转印垫。
- 膜未完全湿润： 遵循预润湿膜的说明。
- 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。
- 封闭不均匀： 孵育皿应足够小，使封闭液能够完全覆盖膜。
- 使用墨水标记膜： 使用墨水对膜进行标记，仅限于印迹膜的低分子量区域。
- iBind卡片损坏： 更换新卡片。应确保膜在卡片上的移动仅限于膜区域内。
- 膜与iBind卡片接触不良： 加入1 mL 1X iBind溶液/iBind荧光检测（FD）溶液后，立即将膜放到iBind卡片上。使用附带的滚轮，确保接触良好。
以下是可能原因和解决方案：
- iBind卡片损坏： 更换新卡片。应确保膜在卡片上的移动仅限于标有“膜”的区域内。
- 膜组在运行前是湿的： 应确保将5 mL 1X iBind溶液/iBind荧光检测（FD）溶液加到iBind卡片的平坦区。不要将溶液加到膜组上。
- iBind溶液/iBind荧光检测（FD）溶液准备不当： 按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）的说明，准备1X iBind溶液/iBind荧光检测（FD）溶液。
以下是可能原因和解决方案：
- 蛋白质上样量过高： 减少上样量或稀释样品浓度。
- 转印效果差或转印不完全： 重复转印。
- 一抗浓度过高： 按照生产商的建议稀释一抗或通过点印迹法确定最佳抗体浓度。
以下是可能原因和解决方案：
- 转印效果差或转印不完全： 重复转印。转印后，对膜进行染色以衡量转印效率。使用阳性对照和/或分子量标记物。
- 膜未完全湿润： 遵循预润湿膜的说明。
- 一抗浓度过低： 按照生产商的建议稀释一抗或通过点印迹法确定最佳抗体浓度。
- 一抗失活： 通过点印迹法确定抗体活性。
- 一抗与抗原的亲和力较低： 获取更高亲和力的一抗。
- 二抗溶液污染： 始终佩戴手套，瓶子在未使用时始终保持拧紧状态。制备试剂时，只使用纯水。
- 目标蛋白跑出凝胶： 应确保凝胶分离范围与待转印蛋白的大小是匹配的。
- 蛋白质保留较差： 应确保凝胶分离范围与待转印蛋白的大小是匹配的。使用具有相关大小蛋白质的分子量标记物。较大的蛋白质需要较长的转印时间，较小的蛋白质所需转印时间较短。使用具有适当结合能力的膜。
- iBind溶液/iBind荧光检测（FD）溶液准备不当： 按照使用手册的说明，准备1X iBind溶液/iBind荧光检测（FD）溶液。
- 向iBind孔中加入溶液时出错： 应确保将溶液加到正确的孔中，并按正确顺序向孔中加液。
- 印迹膜在iBind卡片上的位置不当： 应确保印迹膜上有蛋白质的一面与iBind卡片接触，并放置在标有“膜”的区域。
- 膜组在运行前是湿的： 应确保将5 mL 1X iBind溶液/iBind荧光检测（FD）溶液加到iBind卡片的平坦区。不要将溶液加到膜组上。
- 不同孔中的溶液发生交叉污染： 运行期间，不要移动iBind蛋白印迹处理仪。
- iBind卡片损坏： 更换新卡片。应确保膜在卡片上的移动仅限于标有“膜”的区域内。
- 膜与iBind卡片接触不良： 加入1 mL 1X iBind溶液/iBind荧光检测（FD）溶液后，立即将膜放到iBind卡片上。使用附带的滚轮，确保接触良好。
- 在运行结束前打开设备： 卡片在设备中时，不要打开设备。重新密封卡片上的孔，可导致泄漏。
- 样品制备不当；抗原性减弱或损毁： SDS和还原剂可能干扰一些抗体/抗原亲和力
- 样品太稀： 提高蛋白质样品的浓度，或增加上样量。
- 蛋白质与膜的结合较弱： 转膜液应含10–20%甲醇。
- 曝光时间不足： 对膜进行二次曝光，并延长曝光时间。
- 底物孵育不充分： 应确保每一步都达到指定时间，或在达到可接受的信噪比时，从底物中取出印迹膜。
- 底物污染： 始终佩戴手套，瓶子在未使用时始终保持拧紧状态。制备试剂时，只使用纯水。
- 印迹太旧： 蛋白质会随时间降解。应使用新制备的印迹。
以下是可能原因和解决方案：
- 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。
- 一抗浓度较高： 按照生产商的建议稀释一抗或通过点印迹法确定最佳抗体浓度。
- SDS残留或转印后蛋白质与膜的结合较弱： 根据使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）的指示，遵循免疫检测前的膜准备说明。
- 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。
- iBind溶液/iBind荧光检测（FD）溶液准备不当： 按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）的说明，准备1X iBind溶液/iBind荧光检测（FD）溶液。
以下是可能原因和解决方案：
- 膜未完全湿润：遵循预润湿膜的说明。孵育皿应足够小，使封闭液能够完全覆盖膜，防止膜干燥。注意：如果使用的是PVDF膜，我们建议确保使用甲醇活化，特别是干燥的膜。刚刚结束转印并转移到1X iBind溶液/iBind荧光检测（FD）溶液中的PVDF膜无需活化。
- 膜污染：仅使用干净的新膜。始终佩戴干净的手套并使用镊子来处理膜。
-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。
- 溶液或孵育托盘污染：使用干净的玻璃器皿和纯水制备溶液。使用新的托盘或使用玻璃器皿专用去污剂彻底清洗托盘。用纯水彻底清洗。始终佩戴干净的手套。
- 一抗浓度较高：按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）的说明稀释一抗或通过点印迹法确定最佳抗体浓度。
- 对PVDF膜使用了错误的化学发光底物：应确保使用无增强剂的CDP-Star试剂。
- 印迹膜显色过度：遵循推荐的显色时间，当达到可接受的信噪比时，从底物中取出印迹膜。
- 使用墨水标记膜：使用墨水对膜进行标记，仅限于印迹膜的低分子量区域。
- iBind溶液/iBind荧光检测（FD）溶液准备不当：按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）的说明，准备1XiBind溶液/iBind荧光检测（FD）溶液。
- 向iBind孔中加入溶液时出错：根据使用手册（https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf）第18页的说明，按正确顺序向每孔中加入合适的溶液。
- 印迹膜在iBind卡片上的位置不当：1）将膜置于iBind卡片上的指定膜区域。2）印迹膜上有蛋白质的一面应与iBind卡片接触。3）低分子量区域应与膜组最近。4）膜不应该与膜组接触。
以下是关于降低背景的一些其他技巧：
•如果使用iBlot PVDF膜组，应确认其尚未超过有效期，因为背景会随时间升高。使用iBlot 2 PVDF膜组将有助于降低背景。
• 应确保在加入底物前将印迹膜置于蒸馏水中清洗。不要在PBS或TBS中清洗。
Here are possible causes and solutions:
- Poor or incomplete transfer: Repeat blot.
- Membrane pads are dirty or contaminated: Soak with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- 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.
- Uneven blocking: The incubation dish must be small enough to allow thorough coverage of membrane.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to membrane region.
- Membrane is not in proper contact with the iBind Card: Place the membrane on the iBind Card immediately after adding a 1 mL pool of 1X iBind Solution/ iBind FD Solution. Use the roller provided to ensure proper contact.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane&#!48;.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Solution/ iBind FD Solution is added to the flat region of the iBind Card. Avoid adding solution to the Stack.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Protein is overloaded: Reduce load or dilute concentration of sample.
- Poor or incomplete transfer: Repeat blot.
- Primary antibody is too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Poor or incomplete transfer: Repeat blot. After blotting, stain membrane to measure transfer efficiency. Use positive control and/or molecular weight marker.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- Primary antibody concentration too low: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Inactive primary antibody: Determine activity by performing a dot-blot.
- Low affinity of primary antibody to antigen: Obtain a higher affinity primary antibody.
- Contaminated secondary antibody solution: Wear gloves at all times and keep bottles tightly capped when not in use. Use only purified water when preparing reagents.
- Protein of interest ran off the gel: Match gel separation range to size of protein being transferred.
- Poor retention of proteins: Match gel separation range to size of protein being transferred. Use a molecular weight marker with relevant size proteins. Larger proteins require more transfer time, smaller proteins less. Use membrane with the appropriate binding capacity.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.
- Improper application of solutions to iBind Wells: Ensure that the solutions are added to the correct wells and that the wells are loaded in numerical order.
- Blot improperly placed on iBind Card: Ensure that the protein side of the blot is in contact with the iBind Card and is placed in the region labeled “membrane”.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Solution/ iBind FD Solution is added to the flat region of the iBind Card. Avoid adding solution to the Stack.
- Cross-contamination of solutions in wells: Do not move the iBind Western Device during the run.
- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
- Membrane is not in proper contact with the iBind Card: Place the membrane on the iBind Card immediately after adding a 1 mL pool of 1X iBind Solution/ iBind FD Solution. Use the roller provided to ensure proper contact.
- Device opened prior to completion of run: The device should not be opened once the card has been placed in the device. Re-sealing of the wells on the card can result in leaks.
- Sample improperly prepared; antigenicity weakened or destroyed: SDS and reducing agents may interfere with some antibody/antigen affinities.
- Sample too dilute: Load a higher concentration or amount of protein onto the gel.
- Protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol.
- Insufficient exposure time: Re-expose film for a longer period of time.
- Insufficient substrate incubation: Perform each step for the specified amount of time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Substrate is contaminated: Wear gloves at all times and keep bottles tightly capped when not in use.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Primary antibody too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Insufficient removal of SDS/weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection, as directed in the manual.
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Membrane not completely wet: Follow instructions for prewetting the membrane. Use an incubation dish which is small enough to allow thorough coverage of membrane to prevent drying out.
Note: If PVDF membrane is being used, we recommend making sure that it is activated with methanol, especially if it has dried up. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into 1X iBind Solution/ iBind FD Solution.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Film overexposed or became wet during exposure: Decrease exposure time or allow signal to further decay. Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
- Solutions or incubation tray are contaminated: Use clean glassware and purified water to prepare solutions. Replace or clean the tray thoroughly with a glassware-cleaning detergent. Rinse thoroughly with purified water. Wear clean gloves at all times.
- Concentrated primary antibody used: Follow the instructions provided in the manual ((https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf)) to dilute the primary antibody or determine the optimum concentration by dot blotting.
- Incorrect chemiluminescent substrate used for PVDF: Make sure CDP-Star reagent without enhancer is used.
- Blot is overdeveloped: Follow recommended developing time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.
- Improper application of solutions to iBind Wells: Add the appropriate solutions for each well in the correct numerical order as specified on Page 18 in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).
- Blot improperly placed on iBind Card: 1) Place the membrane in the designated Membrane Region on the iBind Card. 2) The protein side of the blot should be in contact with the iBind Card. 3) The low MW regions should be closest to the Stack. 4) The membrane should not be in contact with the Stack.
Here are some additional tips to reduce background:
- If iBlot PVDF stacks are used, check that they have not expired as background increases with age. Using the iBlot 2 PVDF stacks will help in reducing the background.
- Make sure that the blot is rinsed in distilled water prior to adding the substrate. Do not rinse in PBS or TBS.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.