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查看更多产品信息 Goat anti-Mouse IgM Heavy Chain Secondary Antibody, Alexa Fluor™ 680 - FAQs (A21048)
23 个常见问题解答
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。
- 二抗浓度较高： 按照推荐方法稀释二抗。如果背景仍然很高，但条带强度也很高，则应降低二抗的浓度。
- 一抗浓度较高： 降低一抗浓度。
- 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。
以下是信号弱/无信号的可能原因和解决方案：
- 转印效果差或转印不完全： 检查转印条件或重复转印。使用阳性对照和/或分子量标记物。
- 硝化纤维素膜未完全湿润，或PVDF膜未完全再活化： 按照使用手册第12页的说明，预润湿或再活化膜。
- 二抗浓度过低： 使用推荐的二抗浓度。
- 一抗浓度过低： 将一抗浓度调整为标准免疫印迹检测所需一抗浓度的2倍。如果信号仍然很低并且背景不高，可增加浓度。
- 一抗失活： 通过点印迹法或其他方法确定抗体活性。
- 一抗与抗原的亲和力较低： 获取更高亲和力的一抗。
- 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。
- 印迹太旧： 蛋白质会随时间降解。应使用新制备的印迹。
- 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： 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。使用具有相关大小蛋白质的分子量标记物。使用具有适当结合能力的膜。
- Alexa Fluor 790和680试剂反复冻融： 反复冻融会导致抗体发生不可逆性沉淀。长期保存时，最好在冷冻前将试剂分装到单独的管子中。
以下是可能原因和解决方案：
- 封闭不充分或发生非特异性结合：尝试使用不同的封闭剂或增加封闭剂浓度。一般情况下，使用2%酪蛋白、5%脱脂奶粉或1/2x鱼血清可得到良好的结果。
- 使用BSA封闭膜： 不要使用含BSA的溶液封闭或孵育Alexa Fluor 680和790标记物。对于不能兼容酪蛋白或牛奶（如许多抗磷蛋白抗体）的一抗，应使用鱼血清或含0.5% BSA的溶液进行一抗孵育，然后换成2%酪蛋白或5%脱脂奶粉完成所有其他孵育步骤。
- 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。
- PVDF膜本身具有较高的背景： 改为使用硝化纤维素膜。
- 硝化纤维素膜未完全湿润： 遵循预润湿膜的说明。
- 洗膜不充分： 遵循推荐的洗膜次数。在一些情况下，可能有必要增加洗膜次数和时间。
- 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。
在二抗孵育步骤中，向封闭剂中加入0.1% SDS，以进一步减少非特异性背景染色。使用低荧光PVDF-FL膜代替PVDF膜。
可以，经Alexa Fluor 680和790染色的印迹膜在湿润或干燥状态下均可成像。为了长期保存，我们建议将膜干燥。
所有Alexa Fluor 680和790二抗结合物的供应规格均为0.5 mL 的2 mg/mL储液或1 mg粉末。当工作浓度为5 µg/mL时，可供200张印迹膜染色，每张膜10 mL。
良好的起始工作浓度约为0.5 µg/mL，即2 mg/mL储液按1:4000稀释。最佳浓度范围0.1–1 µg/mL，具体浓度取决于目标蛋白的丰度。
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。
技术上，非远红外Alexa Fluor抗体可用于蛋白质免疫印迹，但是灵敏度比其他检测方法差，所以不推荐使用。印迹膜，特别是PVDF，具有较高的荧光背景，蓝/绿范围内的荧光最强，这会增强噪音并降低灵敏性。封闭蛋白也可能是具有自体荧光的，会增加背景。远红外范围内的印迹膜荧光背景非常低，所以Alexa Fluor 680和790染料具有较高的灵敏度。非远红外Alexa Fluor染料不能制备良好的免疫印迹检测试剂的第二个原因是，非Alexa Fluor 680和790抗体结合物经过优化，可产生较高的免疫荧光（IF）和免疫组化（IHC）信号，并且通常具有较高的染料：抗体标记比例，导致染料与印迹蛋白和膜之间发生大量的非特异性电荷结合，从而增强染色背景并降低信噪比。
Alexa Fluor 680和790二抗结合物具有不同程度的标记，专为免疫印迹检测而优化，因此，它们的结合效率可产生较高的信号和较低的非特异性结合。
Alexa Fluo 680和790二抗试剂的灵敏性与ECL化学发光检测相近。灵敏度在皮克范围内。与基于酶反应的化学发光或发光检测法相比，远红外荧光检测具有很多优势：
•可同时在同一张印迹膜上进行多重检测
•方案简单，对时间不敏感，无需混合试剂
•实验方案可靠，不会产生反应过度或不充分的印迹膜
•光稳定性较高，干燥的印迹膜可归档保存
•使用更常用的紫外或蓝光成像仪，而不是昂贵的化学发光成像仪或滤镜和化学品
•远红外二抗结合物与一抗的目标蛋白直接连接，可产生更清晰的条带，而不会出现酶检测方法产生的蛋白条带边缘扩散现象
Alexa Fluor 680和790结合型二抗的检测灵敏度与相同浓度的Li-COR IRDye 680和800染料相当。
Not likely. With any secondary antibody, you need to consider what other isotypes and species they are cross-adsorbed against. If it is cross-adsorbed against other species or specific chains, cross-reactivity should be minimal or none. Our goat anti-IgM secondary antibodies have been cross-adsorbed against IgG and therefore should exhibit minimal binding to IgG.
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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.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are possible causes and solutions for weak/no signal:
- Poor or incomplete transfer: Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
- Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated: Follow instructions for pre-wetting or reactivating the membrane.
: Secondary antibody concentration too low: Use the recommended secondary antibody concentrations.
- Primary antibody concentration too low: Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
- Inactive primary antibody: Determine activity by performing a dot-blot or other methods.
- Low affinity of primary antibody to antigen: Obtain a higher affinity primary antibody.
- 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.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.
- Protein of interest ran off the gel: Match gel separation range to the size of the protein being transferred.
- Poor retention of proteins: Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
- Alexa Fluor 790 and 680 reagents have been repeatedly frozen: Repeated freeze/thawing can cause antibodies to irreversibly precipitate. For long-term storage, it is best to aliquot into individual use tubes before freezing.
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Here are possible causes and solutions:
- Insufficient blocking or non-specific binding: Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2X fish serum.
- Membrane was blocked with BSA: Do not use BSA-containing solutions for blocking or incubating Alexa Fluor 680 and 790 conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Insufficient washing: Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentration of primary and/or secondary antibody is too high: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
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Add 0.1% SDS to blocker for secondary antibody incubation step to further reduce nonspecific background staining. Use lower fluorescent PVDF-FL membranes rather than PVDF.
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Yes, Alexa Fluor 680 and 790-stained blots can be imaged wet or dried. We recommend drying blots for long-term storage.
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All Alexa Fluor 680 and 790 secondary conjugates come supplied as 0.5 mL of a 2 mg/mL stock or as a 1 mg powder, which is sufficient to stain 200 blots at a working concentration of 0.5 µg/mL and 10 mL/blot.
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A good initial working concentration is ~0.5 µg/mL, which is a 1:4000 dilution of the 2 mg/mL stock. Depending on the abundance of your target, the optimal concentration may be in the range of 0.1-1 µg/mL.
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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).
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Technically, the non-far red Alexa Fluor antibodies could be used on western blots, but they will give very poor sensitivity compared to other detection methods and thus are not recommended. Blot membranes, especially PVDF, have a high fluorescent background, highest in the blue/green range, which increases noise and thus lowers sensitivity. Blocking proteins can also autofluoresce, increasing background. Blot fluorescent background is very low in the far-red range, which is why Alexa Fluor 680 and 790 dyes can obtain high sensitivity. A second reason why non-far red Alexa Fluor dyes do not make good western blot detection reagents is that the non-Alexa Fluor 680 and 790 antibody conjugates are optimized to give high signals for immunofluorescence (IF) and immunohistochemistry (IHC) staining and generally have a high degree of dye:antibody labeling, which can lead to high nonspecific charge-based binding of the dyes to western blotted proteins and the membrane, increasing background staining and thus lowering signal to noise. The Alexa Fluor 680 and 790 secondary antibody conjugates have degrees of labeling that are optimized for western detection, so they have a conjugation efficiency that gives a high signal with lower nonspecific binding.
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Alexa Fluor 680 and 790 secondary antibody reagents have similar sensitivity as ECL chemiluminescent detection. The sensitivity is in the picogram range. Far-red fluorescent detection has a number of advantages over enzyme-based chemiluminescent or chromogenic detection methods:
- Ability to do multiplex detection on the same blot at the same time
- Simple protocol that is not time-sensitive and does not require mixing of reagents
- Reliable protocol that can never give over- or under-developed blots
- High photostability enabling dried blots to be archived
- Uses more commonly available UV or blue light imagers rather than more-expensive chemiluminescent imagers or film and chemicals
- Sharper bands due to the direct linking of the far-red secondary antibody conjugate to the primary antibody target protein, rather than the diffuse edges around a protein band seen with enzyme-based detection methods
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Detection sensitivity with Alexa Fluor 680 and 790 conjugated secondary antibodies is comparable to detection with Li-COR IRDye 680 and 800 dyes used at the same concentration.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.