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查看更多产品信息 WesternDot™ 625 Western Blot Kits - FAQs (W10132, W10142)
75 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。
- WesternDot试剂被冷冻： 在冷冻温度下，Qdot探针会发生不可逆性聚集。
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WesternDot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。
如果是大量沉淀，则可能表示试剂被冷冻过，应丢弃。在冷冻温度下，Qdot探针将发生不可逆性聚集。保存于2-8℃时，形成少量沉淀属于正常情况。我们建议每次使用时将WesternDot试剂在微型离心机中短暂离心，除去保存期间形成的所有少量沉淀，仅使用上清液。
细胞裂解物样品中的许多内源性生物素化蛋白也会被链霉亲和素WesternDot标记物检测到。将对照印迹膜放在链霉亲和素WesternDot标记物中孵育，而不经过一抗和二抗孵育，可确定内源性生物素化蛋白。这些内源性生物素化蛋白条带在所有含有相同类型细胞裂解物的泳道中都是一致的，可将其作为上样内参或在免疫检测前使用内源性生物素染色封闭剂（货号E21390）将其封闭。
结合在干燥PVDF膜上的WesternDot信号会衰减。为了将染色的印迹膜存档或在超过24小时以后成像，我们推荐使用硝化纤维素膜。
许多因素可降低信号强度。其中一个主要因素是一抗。不同来源的一抗对目标抗原的亲和力差别很大。如果得到的信号较弱，可尝试使用不同供应商和/或不同宿主来源的一抗，这将有助于改善信号强度。
硝化纤维素和PVDF膜均可使用。为获得更好的结果，我们建议使用硝化纤维素膜，从而将膜自体荧光产生的背景降至最低。
从收到之日起，WesternDot检测试剂可在2-8℃下稳定保存至少6个月，我们不建议冷冻保存。
可能可以，但尚未被验证。应使用我们的任意一种链霉亲和素标记的Qdot探针（如，我们的Qdot 625链霉亲和素结合物，货号Q22063），利用Northern或Southern印迹杂交实验，对生物素化探针进行检测。
与ECL检测相比，WesternDot检测试剂和远红外染料的灵敏度与其相近或更好。两种试剂均使用相似的染色方案，并且在干燥的印迹膜上可被归档保存。同时，WesternDot试剂和远红外染料都是直接与一抗目标蛋白连接，因此，染色蛋白条带的分辨率非常清晰，而不会出现基于酶/底物的化学发光和发光检测法产生的蛋白条带边缘扩散现象。与远红外荧光染料相比，WesternDot试剂的优势是紫外-蓝光激发波长与发射波长非常远，因此，实际上不存在来自于印迹膜或封闭蛋白的自体荧光背景，可获得良好的信噪比和对比度非常高的图像。远红外荧光染料的激发波长与发射波长很近，所以，会产生一些来自于印迹膜或封闭蛋白的自体荧光背景。WesternDot检测试剂的光稳定性比荧光染料更强，染色或操作过程无需避光。WesternDot检测试剂只需大多数凝胶和印迹膜成像仪常用的紫外或蓝光激发光源，无需购买特殊的成像仪。WesternDot 625试剂与溴化乙锭染色凝胶使用相同的激发光源和发射光滤镜。
不建议这样做，因为，用于制备WesternDot结合物的Qdot探针显著大于IgG。Qdot探针约为1.5X10^6 — 2X10^6 Da（与IgM大小相近），因此，在凝胶基质中的渗透率远远低于无标记的抗体或者HRP或碱性磷酸酶标记的抗体。该试剂可能可以用于经过一些优化的比例非常低的丙烯酰胺凝胶，但是灵敏性会低于印迹膜。
不能，Li-COROdyssey成像仪的激发波长为685 nm和785 nm。为获得最佳灵敏度，您需要使用带有紫外或蓝光激发器的仪器。WesternDot试剂在它们的发射峰附近激发较弱，所以，您可能会看到一些信号，但灵敏性远远低于远红外荧光染料。
经WesternDot结合物染色的印迹膜成像时，应将蛋白质一面朝向光源。所以，在紫外透照仪上，印迹膜应正面朝下。
使用紫外范围激发可产生最亮的信号和最低的灵敏度，但是，也可使用带有蓝光光源（如，450 nm或473/488 nm）的成像仪。
不需要，湿润或干燥的印迹膜均可用于成像。为维持信号的长期稳定，印迹膜应保持干燥状态。
WesternDot试剂具有光稳定性，因此，干印迹膜可长期保存而无信号强度损失。
WesternDot试剂具有光稳定性，因此可进行多次成像而无信号强度损伤。
任何带有紫外或蓝光激发器以及溴化乙锭滤镜的基础凝胶或印迹膜成像仪，都可用于WesternDot 625结合物的单色检测。带有紫外或蓝光底座或橙色滤镜的E-Gel成像仪，可用于对WesternDot 585或WesternDot 625结合物染色的印迹膜进行单色检测。对于双色或三色多重检测，GE Healthcare ImageQuant LAS 4000含有适用于WesternDot585、625、655和800结合物成像的滤镜套装。关于所有WesternDot试剂的其他特殊成像仪和滤镜组合，请参见Western Dot二抗结合物使用手册中的表2（https://tools.thermofisher.com/content/sfs/manuals/westerndot_secondary_ab_conj_man.pdf）。
可以，蛋白质免疫印迹处理设备适用于WesternDot试剂。
可以，使用WesternDot试剂和WesternBreeze CDP-Star、WesternBreeze BCIP/NBT和ECL试剂可进行多重检测。WesternBreeze CDP-Star是该应用的首选检测试剂。依次在一抗和二抗溶液中共同孵育，然后进行WesternDot检测。WesternDot成像使用的紫外激发和发射光滤镜无法检测到CDP-Star化学发光信号，因此，在CDP-Star底物孵育前或者之后在湿润或干燥的印迹膜上均可对WesternDot信号进行成像。WesternBreeze BCIP/NBT和ECL信号可在WesternDot成像中被检测到，因此，在加入这些检测试剂前，必须在湿印迹膜上首先对WesternDot信号进行成像。可在此处 (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/support/bioprobes/bioprobes-63.par.51550.file.dat/bioprobes-63-westerndot.pdf)查找更详细的操作步骤。
可以，可使用试剂将印迹膜剥离并再次检测。一般来说，WesternDot结合物比标准品染料或蛋白标记的二抗结合物更易去除，一抗最难去除。您应使用能够去除一抗的剥离液。我们利用Restore和Restore Plus剥离液并按照它们的操作步骤在室温下进行剥离，获得了良好的结果。在50℃加热，可完全去除一抗。WesternDot试剂具有多重检测能力，使用合适的可兼容性一抗和WesternDot试剂，即可对多种蛋白同时进行检测，因此，不再需要剥离和再次检测。
多重蛋白检测过程与单一蛋白检测相同。将蛋白质转印到PVDF膜或硝化纤维素膜上并封闭后，使用各目标蛋白的一抗混合液孵育印迹膜，然后使用各一抗的WesternDot二抗结合物混合物进行检测。例如，对Akt、磷酸化Akt和GAPDH进行三色多重检测，则使用兔抗Akt抗体、鼠抗磷酸化Akt和鸡抗GAPDH抗体的混合液孵育印迹膜，然后使用WesternDot 605山羊抗兔IgG、WesternDot 655山羊抗鸡完整IgG和WesternDot 800山羊抗鼠IgG的混合物进行检测。
不能，WesternDot染色液应在配制后立即使用，并且只能使用一次。Qdot探针稀释到缓冲液中后，不能长期保持稳定。
不需要。Qdot探针在正常室内光照条件下很稳定，因此，印迹膜孵育和干燥无需避光。
吐温20是免疫印迹缓冲液配方中常用的去污剂。对WesternDot二级偶联物来说，我们发现缓冲液中使用吐温20对硝化纤维膜印记是适用的，但不是理想结果的必要条件。因为吐温20会导致PVDF膜的高背景，所以不建议在WesternDot二级偶联物和PVDF膜联用时使用吐温20。如果使用了PVDF膜，应该在所有缓冲液配方中省略吐温20。
为获得最佳结果，应参考供应商的建议孵育时间，或对孵育时间进行优化。一般来说，一抗可在室温下孵育1小时，或在4℃孵育过夜。对于WesternDot二抗结合物，我们建议在室温下孵育1小时。
WesternDot二抗结合物的建议稀释倍数为1:500。使用该浓度，每管WesternDot二抗结合物可供25张免疫印迹膜使用。
不同的一抗具有不同的理想工作浓度。为获得最佳结果，应参考抗体供应商的建议条件，或在使用前对一抗的工作浓度进行优化。
任何一种缓冲液配方都不能完全适用于所有蛋白质/抗体：
- 含2%酪蛋白、5%脱脂干奶粉或1/2X鱼血清（如Fish Serum Blocking Buffer）的1x PBS/TBS溶液适用于大多数目标蛋白和抗体组合。
- 不要使用含BSA的溶液封闭或孵育WesternDot结合物，否则可能导致高背景和/或信号降低。
- 对于不兼容酪蛋白或牛奶的一抗（如，许多抗磷蛋白抗体），应使用鱼血清或含0.5%BSA的溶液。如果使用BSA，则仅能用于一抗孵育；在所有其他步骤中，应使用2%酪蛋白、5%脱脂奶粉或1/2X鱼血清。
对于蛋白质免疫印迹，将蛋白质从SDS-PAGE凝胶转印到硝化纤维素或PVDF膜上后，立即使用20 mL纯水洗膜2次，每次5分钟，从而除去部分凝胶和转移缓冲液成分以及弱结合蛋白质。随后，膜可立即用于WesternDot免疫检测实验方案。
或者，将洗过的膜放在一张干净的滤纸上，在微热气流或红外灯下蒸干。适当干燥的膜可在4℃的密封容器内保存7天，保存时间取决于抗原。经水洗和干燥的硝化纤维素膜可立即用于WesternDot免疫检测实验方案。但是，经水洗和干燥的PVDF膜需要在甲醇中再润湿，随后用20 mL水洗涤2次，每次5分钟，最后用于WesternDot免疫检测实验方案。
对于非变性PAGE蛋白质免疫印迹膜，建议在所有洗涤步骤前实施干燥步骤，改善蛋白质与膜的结合。干燥后，应使用20 mL水将硝化纤维素膜洗涤2次，每次5分钟，然后再用于WesternDot免疫检测实验方案。干燥的PVDF膜需要在甲醇中再润湿，随后用20 mL水洗涤2次，每次5分钟，最后用于WesternDot免疫检测实验方案。
所有独立的WesternDot试剂均可供25张小型凝胶印迹膜染色。WesternDot625山羊抗鼠和山羊抗兔试剂盒（货号W10132和W10142）所含试剂可供20张小型凝胶印迹膜染色。
可以，它们的货号分别是B2763和B2770。
可以，Qdot 625链霉亲和素结合物（货号Q22063，50 μL或货号A10196，200 µL）可以单独购买。
与基于酶反应的化学发光或显色检测法相比，WesternDot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
•对时间不敏感的简单方案，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
•WesternDot试剂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
与ECL检测相比，WesternDot试剂的灵敏度与其相近或稍高。灵敏度在皮克范围内。
WesternDot试剂是畅销型二抗和精选型单克隆一抗的完整IgG（H+L）结合物，或红外或远红外荧光Qdot探针、Qdot 585、Qdot 625、Qdot 655和Qdot 800的链霉亲和素结合物，可用于蛋白质免疫印迹分析。Qdot探针的发射范围较窄且对称，与其他发光颜色的重叠极小，向相邻检测通道的渗透更少，因此可同时利用多种颜色。使用凝胶和印迹膜成像仪常用的任何紫外或蓝光光源，都能实现同等的最佳激发。此外，Qdot探针的光稳定性极强，可多次成像，并且干燥的印迹膜可保存数月而荧光信号损失极小。
若想获得关于Qdot技术的更多信息，请查看以下链接：
http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/Qdot/technology-overview.html
http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/Qdot/technology-overview.html
Any dye in the 625 nm emission range should work. For example, you can use the WesternDot 625 Western Blot Kit.
We also offer our White-Light Conversion Screen (Cat. No. 4473061), which converts blue light emitted by the blue-light transilluminator or UV light emitted by the UV-light transilluminator to white light. This conversion screen is compatible with multiple protein stains including SimplyBlue SafeStain, SilverQuest silver, and Coomassie blue stains.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis 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.
- 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 antige: 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.
- WesternDot reagents have been frozen: Qdot probes will irreversibly aggregate at freezing temperatures
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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 WesternDot 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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
If there is a large amount of precipitate, it likely indicates that the reagent has been frozen and should be discarded. Qdot probes will irreversibly aggregate at freezing temperatures. The formation of a small amount of precipitate during storage at 2-8 degrees C is normal. We recommend spinning the WesternDot reagent tube down briefly in a microcentrifuge with every use to remove any minor precipitate that has formed during storage and use only the supernatant.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
There are a number of endogenous biotinylated proteins in cell lysate samples that will also be detected with streptavidin WesternDot conjugates. Endogenous biotinylated proteins can be confirmed by incubating a control blot in only the streptavidin WesternDot conjugate without the primary and secondary antibody. These endogenous biotinylated protein bands will be consistent in every lane containing the same cell lysate type and can serve as internal loading controls or can be blocked prior to western detection using the reagents in the Endogenous Biotin Blocking Kit (Cat. No. E21390).
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WesternDot signals can fade when bound to dried PVDF membranes. For archiving stained blots or imaging after more than 24 hours, we recommend using nitrocellulose membranes.
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Many factors may reduce signal intensity. One major factor is the primary antibody. Primary antibodies from different sources can have very different affinities to the target antigen. If weak signal is observed, try a primary antibody from a different vendor and /or from different host species, as this can help to improve signal intensity.
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Both nitrocellulose and PVDF membrane can be used. For better results, we recommend using nitrocellulose membrane to minimize background from autofluorescence.
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WesternDot detection reagents are stable for at least 6 months from the date of receipt when stored at 2-8 degrees C, We do not recommend freezing them.
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This may be possible, but has not been validated. A biotinylated probe should be detected with any of our streptavidin labeled Qdot probes (such as our Qdot 625 streptavidin conjugate, Cat. No. Q22063) using standard northern or Southern blotting protocols.
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The sensitivity of WesternDot detection reagents and the far-red dyes are all similar to or better than ECL detection. Both types of reagents use similar staining protocols and can be archived on dried blots. Also, WesternDot reagents and far-red dyes are both directly linked to the primary antibody target protein, so the resolution of the stained protein band is very sharp, rather than the diffuse edges around a protein band that is seen with chemiluminescent and chromogenic detection via an enzyme/substrate. The advantage of the WesternDot reagents over far-red fluorescent dyes is that UV-blue excitation wavelengths are so far removed from the emission wavelengths that there is virtually no autofluorescence background from the blot membrane or blocking proteins, giving much better signal to noise and thus very high contrast images. Far-red fluorescent dyes are excited close to their emission wavelengths, so there will be some autofluorescence background from the blot membrane and blocking proteins. WesternDot detection reagents are much more photostable than fluorescent dyes and do not require protection from light during staining or handling. WesternDot detection reagents require a UV or blue light excitation source commonly available on most gel and blot imagers and do not require the purchase of a specialized imager. WesternDot 625 reagents use the same excitation sources and emission filters as ethidium bromide-stained gels.
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This is not recommended, as Qdot probes used to make the WesternDot conjugates are significantly larger than IgGs. Qdot probes are estimated to be about 1.5-2 million Da (similar in size to an IgM), so permeability through the gel matrix is going to be much lower than for an unlabeled antibody or even an HRP- or alkaline phosphatase-labeled antibody. It may be possible in a very low percent acrylamide gel with some optimization, but sensitivity will be less than on a membrane.
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No, the Li-COR Odyssey imagers use excitation wavelengths of 685 nm and 785 nm. For optimal sensitivity, you need to use an instrument that has UV or blue light excitation. WesternDot reagents have weak excitation near their emission peak, so you may see some signal, but sensitivity will be much lower than for far-red fluorescent dyes.
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When imaging a WesternDot conjugate-stained blot, the blot should be placed with the protein side toward the light source. On a UV transilluminator, this means that the blot should be placed face down.
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The brightest signal and lowest sensitivity are obtained when exciting in the UV range, but imagers equipped with a blue light source (e.g., 450 nm or 473/488 nm) can also be used.
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No, blots can be imaged wet or dried. For long-term stability of signal, blots should be kept dry.
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The photostability of WesternDot reagents enables long-term storage of dried blots without loss of signal intensity.
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The photostability of WesternDot reagents enables taking multiple images without loss of signal intensity.
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Any basic gel or blot imager with UV or blue light excitation and an ethidium bromide filter can be used with for single-color detection of WesternDot 625 conjugates. The E-Gel imager with the UV or blue light base and the orange filter can be used for detecting single color WesternDot 585 or WesternDot 625 conjugate-stained blots. For two or three-color multiplexing, a GE Healthcare ImageQuant LAS 4000 includes filter sets suitable for imaging WesternDot conjugates in the 585, 625, 655, and 800 colors. Other specific imager and filter combinations for all the WesternDot reagents can be found in Table 2 of the Western Dot Secondary Antibody Conjugates manual (http://tools.thermofisher.com/content/sfs/manuals/westerndot_secondary_ab_conj_man.pdf).
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Yes, western blot processing instruments work well with WesternDot reagents.
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Yes, it is possible to multiplex detection with WesternDot reagents and WesternBreeze CDP-Star substrate, WesternBreeze BCIP/NBT and ECL reagents. WesternBreeze CDP-Star is the preferred detection reagent for this application. Simply co-incubate in the primary antibodies and then in the secondary antibodies following the WesternDot detection protocol. The CDP-Star chemiluminescent signal is not detected with UV excitation and emission filters used for WesternDot imaging, so the WesternDot signal can be imaged before CDP-Star substrate incubation or afterwards on wet or dried blots. WesternBreeze BCIP/NBT and ECL signals will be detected in the WesternDot image, so the WesternDot signal must be imaged first on a wet blot before addition of these detection reagents. Here is a link to a more detailed protocol (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/support/bioprobes/bioprobes-63.par.51550.file.dat/bioprobes-63-westerndot.pdf).
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Yes, blots can be stripped and reprobed with WesternDot reagents. We found that in general, the WesternDot conjugate was removed easier than standard dye or protein-labeled secondary conjugates and that the primary antibody was the hardest to remove. You should be able to use any stripping solution that is strong enough to remove the primary antibody. We have obtained good results with Restore and Restore Plus stripping buffers following their protocol at room temperature. It may take heating at 50 degrees C to fully remove the primary well. The multiplexing capability of WesternDot reagents enables simultaneous detection of multiple proteins using appropriately compatible primary antibodies and WesternDot reagents, so that stripping and reprobing is no longer required.
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The process for multiplexed protein detection is the same as for single protein detection. After transferring proteins onto PVDF or nitrocellulose membranes and blocking, incubate the blot in a mixture of primary antibody solution for each protein target followed by detection with a mixture of WesternDot secondary antibody conjugates against each primary antibody. For example, to do a three-color multiplexing of Akt, phospho-Akt and GAPDH, incubate the blot in a mixture solution of rabbit anti Akt antibody, mouse anti phospho-Akt and chicken anti GAPDH, followed by detection with a mixture of WesternDot 605 goat anti rabbit IgG, WesternDot 655 goat anti chicken whole IgG and WesternDot 800 goat anti mouse IgG.
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No, the WesternDot staining solution should be used as soon as it is prepared and can only be used once. Qdot probes are not stable long-term diluted in buffer.
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No, it is not necessary. Qdot probes are photostable under normal room light illumination, so blots can be incubated and dried without covering to protect from light.
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Tween 20 is a detergent commonly used in western blot buffer formulations. For WesternDot secondary conjugates, we found the use of Tween 20 in buffers to be suitable for nitrocellulose membrane blots, but not necessary for desirable results. Because Tween 20 leads to high background on PVDF membrane, it is not recommended for use with WesternDot secondary conjugates in combination with PVDF. If PVDF membranes are used, Tween 20 should be omitted from all the buffer formulations.
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For best results, refer to the vendor's suggested incubation time, or optimize incubation time. Generally, the primary incubation is carried out for one hour at room temperature, or overnight at 4 degrees C. We recommend an incubation time of 1 hour at room temperature for WesternDot secondary antibody conjugates.
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The recommended WesternDot secondary antibody conjugate working dilution is 1:500. Each vial of WesternDot secondary antibody conjugate contains sufficient material for twenty-five western blots at this working dilution.
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The ideal working concentration can vary between different primary antibodies. For best results, one should refer to the antibody vendor's suggested conditions, or optimize the working concentration for a primary antibody prior to use.
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No single buffer formulation works best for every protein/antibody:
- 2% casein, 5% non-fat dry milk, or 1/2X fish serum (e.g., Fish Serum Blocking Buffer) in 1x PBS/TBS work well for most target protein and antibody combinations.
- Do not use BSA-containing solutions for blocking or incubating WesternDot conjugates as their use may cause high background and/or reduced signal.
For primary antibodies that are incompatible with casein or milk (e.g., many antiphosphoprotein antibodies), use fish serum or a 0.5% BSA-containing solution.
If using BSA, use for the primary antibody incubation only; in all other steps, use 2% casein, 5% non-fat milk or 1/2X fish serum.
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For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternDot immunodetection protocol.
Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternDot immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.
For Native-PAGE Western blots, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 minutes before proceeding to the WesternDot immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.
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All standalone WesternDot reagents are provided with sufficient material to stain 25 Mini gel blots. The WesternDot625 Goat anti-mouse and goat anti-rabbit kits (Cat. Nos. W10132 and W10142) are provided with sufficient reagents to stain 20 Mini gel blots.
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Yes, they can be purchased as Cat. Nos. B2763 and B2770, respectively.
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Yes, it can be purchased as Qdot 625 Streptavidin Conjugate (Cat. No. Q22063, 50 µL or Cat. No. A10196, 200 µL).
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WesternDot 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 WesternDot reagents 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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WesternDot reagents have similar or slightly better sensitivity compared to ECL detection. The sensitivity is in the picogram range.
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WesternDot reagents are whole IgG (H+L) conjugates of our popular secondary antibodies and select monoclonal primary antibodies, or streptavidin conjugates of our red and far-red fluorescent Qdot probes, Qdot 585, Qdot 625, Qdot 655 and Qdot 800, for western blotting applications. Qdot probes have narrow and symmetrical emissions, minimizing overlap with other emission colors, producing less bleed through into adjacent detection channels and allowing multiple colors to be used simultaneously. They are equally optimally excited with any UV or blue light sources commonly available on gel and blot imagers. Furthermore, Qdot probes are extremely photostable, making it possible to take multiple images and store dried blots for months with minimal loss of fluorescent signal.
For more information on our Qdot technology, see the following link:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/qdot-nanocrystal-technology.html
http://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/technology-overview.html
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