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View additional product information for Qdot™ Streptavidin Sampler Kit - FAQs (Q10151MP)
58 product FAQs found
以下是我们的建议：
•使用我们偶联了明亮的、光稳定性Alexa Fluor 染料的二抗。每个荧光二抗IgG分子上标记有2-8个荧光基团，每个一抗含有三个潜在的二抗结合位点，能够提供大约10-20荧光团/抗体的信号放大等级。
•此外，也可采用生物素结合的二抗以及荧光素-链霉亲和素偶联物检测一抗，或者采用链霉亲和素桥连Qdot 生物素等生物素结合的报告载体来检测一抗。尽管额外的孵育和内源性生物素封闭步骤会增加处理时间，但链霉亲和素标记也会提高检测灵敏度。
•对于低丰度的靶标，为得到最佳信噪比，可能要对信号进行放大。酪胺信号放大技术（TSA）是一种酶介导的检测方法，其利用辣根过氧化物酶（HRP）的催化活性产生具备反应活性的荧光团标记酪胺自由基。这些短寿命的酪胺自由基共价结合到相互作用位点临近的残基上，在HRP-靶相互作用位点上产生放大的荧光信号。
•如需提升快速漂白染料的检测灵敏度，我们的SlowFade Diamond或ProLong Diamond 抗猝灭试剂已经证实可增加光稳定性，降低固定细胞、组织和无细胞制备物中的初始荧光淬灭。
•请访问此网页（https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/guide-to-immunocytochemistry.html）了解进一步的优化的要点。
放大抗体检测效果的常用方法是生物素-链霉亲和素检测，使生物素化的二抗结合偶联染料的链霉亲和素。该方法能够将信号放大约2-8倍，但在此之前必须先封闭内源性生物素。另一种方法是使用酪胺信号放大技术，将辣根过氧化物酶偶联物与染料标记的酪胺配合使用。该方法能够将信号放大约10-20倍，但在此之前必须先封闭内源性过氧化物酶。最后一种方法是使用Qdot纳米晶体抗体或链霉亲和素偶联物，根据Qdot颜色，该方法可产生高于标准有机染料偶联物40倍的信号。
部分建议如下：
•使用Qdot 孵育缓冲液（货号Q20001MP）
内附的缓冲液具有特殊配方，在大多数使用Qdot链霉亲和素偶联物免疫标记应用中，均可提高信噪比。其它缓冲液可能会导致更多的不均匀着色，并且尤其可能增加背景着色。然而，一些特别的应用可能会需要其他缓冲液条件。请参见实验方案“使用Qdot链霉亲和素偶联物进行双重标记”（Double-labeling Using Qdot Streptavidin conjugates.）。
•确定样品是否含有高水平的内源性生物素。对样本进行亲和素-生物素预封闭
如果使用Qdot孵育缓冲液却依然得到较高的非特异性背景信号，则可能必须检查操作流程中的其他步骤。用BSA或正常动物血清封闭样品可以降低抗体和Qdot链霉亲和素偶联物的非特异性结合。最好的做法是使用封闭缓冲液稀释您的一抗和二抗。另外，肝脏和肾脏等组织的切片可能含有内源性生物素，其可能产生非特异性信号。内源性生物素能够用亲和素/生物素封闭试剂盒（货号E21390）封闭。
•背景中出现染色颗粒或块状荧光物质
随着时间推移，Qdot孵育缓冲液中的BSA偶尔会轻微聚集。在使用Qdot链霉亲和素偶联物标记样品前必须移除这些聚集物。添加到样品前，可通过离心将孵育混合液中的聚集物沉淀下来。将样品在台式离心机（Eppendorf 5415）中5,000 x g离心2分钟来完成此过程。在将孵育混合液添加到样品中前，也可将其通过0.2 µm的滤膜离心柱以去除微观沉淀。如果你使用Qdot孵育缓冲液之外的其它缓冲液，往往会导致孵育缓冲液中的NaCl或其他盐浓度过高，可能很难通过过滤法解决。在这种情况下，须减少整体盐浓度。
•优化生物素化的二抗工作浓度
调节染色过程中所用的生物素化抗体水平，能够帮助优化特异性信号。为实现特异性标记，必须要提供较高水平的生物素化抗体，但抗体水平过高又会导致抗体与样品发生非特异性结合。Qdot链霉亲和素偶联物会连接到非特异性结合的生物素化抗体上，导致较高的背景染色。
•优化Qdot链霉亲和素偶联物的工作浓度
正如免疫标记应用中，滴定一抗和二抗是维持最佳的特异信号的必备条件，应该对每个种偶联物都应该优化其工作浓度。总的来说，等于或轻微低于饱和浓度的情况下可获得最佳的信噪比，相反，当高于饱和浓度时将会导致背景信号升高。
以下为我们的一些建议：
•确认成像/检测设置是否适当
确保使用合适的滤镜组来检测信号。请查阅Qdot生物素使用手册的表1来获取适当的滤镜信息。
•用替代光源检查Qdot偶联物是否发荧光。
Qdot偶联物通常会在手持式紫外灯（长波长，例如用于显示琼脂糖凝胶上溴化乙锭的这类紫外灯）下发出强的荧光。尽管在任何测试过的储存条件下我们暂未发现这些Qdot产品有明显的荧光损失，但我们毕竟没法逐个考察所有储存条件。如果Qdot产品在长波长UV激发下没有出现荧光，请联系techsupport@qdots.com获取技术支持。利用显微镜，可以进行光斑检测：在一块干净的载玻片上（无盖玻片）滴上一小滴（2到5 μL）的量子点溶液，并在适当的滤片和低放大倍数下检测。
•确认一抗的特异性和滴定量
确保抗体可以识别预定的靶标，同时确保有足够的一抗能结合到靶点。可以通过ELISA法捕获靶标抗原或根据实验室手册（例如免疫学最新手册）所述的其他技术来进行验证。
•对于Qdot链霉亲和素偶联物，请确认抗体具有生物素化修饰
确保抗体被有效生物素化修饰。另外，可能需要单独调节实验所用的一抗和二抗浓度来优化信号并降低背景信号。
•PAP笔墨水可能会淬灭信号
使用替代方法隔离玻片上的靶向区域。如果您的方案需要用到PAP笔，我们推荐采用Vector Labs的ImmEdge免疫组化笔（货号H-4000）
在3,000 rpm下离心ITK Qdot纳米晶3-5分钟，可以从上清中去除白色沉淀，之后就可以直接使用上清了。
有机ITK Qdot纳米晶中有一定几率会析出白色沉淀。ITK Qdot纳米晶有时候会含有呈白色沉淀状的杂质。
闪烁是量子点固有的性质；实际上，所有的单一发冷光的分子都会闪烁，包括有机染料。由于其亮度和光稳定性，Qdot纳米晶的闪烁显得更加明显。激发能量越高，Qdot纳米晶闪烁甚至更快。使用Beta-巯基乙醇可以减少闪烁。
选择合适的封固液对保持Qdot纳米晶荧光性能至关重要。根据我们的研究，Qdot纳米晶适合用下列封固液：
•HistoMount封固液（货号00-8030），非常适合长期保存
•Cytoseal 60封固液
•Clarion封固液
•大多数基于聚乙烯醇的封固液（有限储存时间，<数周）
•基于水的封固液（有限储存时间，<1周）
•至多50%的甘油 （有限储存时间，<1周）
注：我们不推荐ProLong封固液与Qdot 纳米晶一同使用，因其会导致Qdot发生荧光淬灭。
冷冻会导致产品聚集。一旦聚集后就不能重新分散到溶液内。
一旦产品形成聚集，就无法重新分散到溶液内。我们建议重新购买。
在正常的储存过程中可能偶然会观察到少量Qdot纳米晶聚集。为了在使用前去除这些聚合物，我们建议在2,000 x g下离心1分钟。仅吸取上层清液并避免吸到沉淀。根据我们的经验，沉淀聚集物通常仅会导致10%以下的产品损耗。
我们没有系统地研究纳米晶的能量转移性质，尽管纳米晶可能作为能量转移的供体和受体。我们已经研究了通过双生物素交联剂相互偶联的Qdot 605链霉亲和素偶联物的荧光性，且发现任何浓度的生物素交联剂都不会影响交联后的纳米晶的发射强度。这些结果表明Qdot偶联物粒子间的淬灭可以忽略。
Qdot产品含有无机晶体形式的镉和硒（大颗粒中含有碲）。对于处理这些材料，我们只能建议您按照所有合适的当地的、州的和联邦条例来处理这些材料。关于这些材料成分的更多信息，查询材料安全数据登记表（Material Safety Data Sheet）。
我们没有研究过Qdot纳米晶的毒性。这种材料是以2 mM总Cd浓度的溶液提供的。我们已经在多种活细胞的体外标记实验中证明这些材料的用途，但是还没有研究这些材料对人类、动物、或培养细胞毒性的系统数据。
Qdot偶联物在非优化的缓冲液中会与样本发生更高程度的非特异性结合。我们在多种不同的缓冲液中，包括TBS、PBS、RPMI培养基等，都可获得成功的染色结果，但是发现在孵育缓冲液中的染色结果是最稳定的。
Qdot 605链霉亲和素偶联物在一系列不同pH的缓冲液中都有稳定的发射峰。在工作浓度下，这些材料在pH 6-9 （超出此范围没有研究）范围内的Tris、HEPES,、磷酸盐和硼酸盐缓冲液中，量子产率和胶体分布都非常稳定。工作浓度下，Qdot 605链霉亲和素偶联物在高达200 mM的NaCl缓冲液中是稳定的和非聚集的。在稀释工作液中，高浓度盐可能会导致微观沉淀，但不会引起材料大量沉淀。另外，若干表面活性剂和添加剂例如Tween 20、Triton X-100、Pluronic F-68、NDSB-201和EDTA等，在0.05%浓度时不会对荧光造成影响。与此相反，发现明胶和葡聚糖硫酸酯在0.05%浓度都可促进Qdot 605链霉亲和素偶联物聚集，因此在标记应用中应该避免使用。总的来说，我们建议以运输的浓度来储存Qdot 605链霉亲和素偶联物而不是高度稀释后保存。在稀释工作液中储存材料超过一段时间可能会导致性能下降。尽管我们并没有在多种缓冲液中表征其它Qdot偶联物的稳定性，我们预期稳定性相似。
偶联到一个Qdot纳米晶上的分子的数量取决于偶联过程中使用的纳米晶与偶联分子的比率，Qdot纳米晶上可用的结合位点数量，以及Qdot纳米晶和目标分子的大小。总的来说，每个Qdot纳米晶上含有2-3个抗体，4-5个生物素分子，6-8个链霉亲和素分子。
ITK Qdot纳米晶使用第一代Qdot产品中外层聚合物的经典组成；除了氨基-PEG产品，外层聚合物不含有PEG。标准Qdot纳米晶的外层聚合物含有PEG。
每个Qdot ITK纳米晶大约有80–100官能团。我们使用一种免疫吸附测定方法来测定每种偶联物的EC50。
可以，您可以使用标准滤光片来观察Qdot纳米晶；可以使用低于发射波长的任何波长激发它们。切记，激发光波长越短，Qdot纳米晶发出的荧光越亮。
Qdot纳米晶不会像化学染料那样光漂白或褪色，不需要使用抗淬灭剂。根据我们的研究，Qdot纳米晶适合用下列封固液：
•HistoMount封固液（货号：00-8030），非常适合长期保存
•Cytoseal 60封固液
•Clarion封固液
•大多数基于聚乙烯醇的封固液（有限储存时间，<数周）
•基于水的封固液（有限储存时间，<1周）
•至多50%的甘油（有限储存时间，<1周）
提示：我们不推荐ProLong封固液与Qdot纳米晶一同使用。
亲水性的Qdot纳米晶在pH 8.3–9.0的硼酸盐缓冲液中储存和运输，而有机Qdot纳米晶则在癸烷中储存和运输。我们研究过Qdot纳米晶在多种不同溶剂中的稳定性，更多信息详见溶剂稳定性（https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/qdot-reg--nanocrystal0.html）。
当在4℃下存储时，Qdot纳米晶可稳定6个月。由于可能发生聚集，Qdot纳米晶绝对不能冷冻。关于最高360°C暴露温度的信息见温度稳定性（https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/qdot-reg--nanocrystal0.html）。
Qdot纳米晶在pH6–9的范围内最稳定，最低至pH 5。由于可能存在自聚集，Qdot纳米晶不能在pH > 9的条件下使用，并且不能在pH < 4的条件下使用，否则聚合物和暴露的核/壳就会开始分离。关于更多详情和pH推荐范围，详见Qdot 纳米晶的pH范围（https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/qdot-reg--nanocrystal0.html）。
你可以在两种FRET情况下使用Qdot纳米晶：
•Qdot纳米晶作为供体而荧光染料作为受体
•镧系元素（铽、铕等）作为供体而Qdot纳米晶作为受体
注意：不得在FRET实验中同时将Qdot纳米晶作为供体和受体。
我们提供氨基（PEG）、羧基和链霉亲和素官能化的Qdot Innovator’s Tool Kit ITK 纳米晶用于偶联自己感兴趣的蛋白或其它生物分子。其中氨基（PEG）衍生形式的纳米晶可以与异硫氰酸盐和琥珀酰亚胺酯偶联，或使用水溶的碳化二亚胺结合羧酸。羧基衍生形式的纳米晶能够结合到蛋白质和修饰的寡核苷酸的氨基基团上。链霉亲和素衍生形式的纳米晶则能够结合生物素化的偶联物来形成稳定的标记复合物。
Qdot纳米晶及其生物偶联物非常适合需要长时间光稳定性、单一激发、多色分析的实验。部分应用示例包括：
•流式细胞术
•细胞和组织染色
•细胞示踪
•WesternDot蛋白免疫印记
•活体成像
相比于传统的荧光染料，Qdot纳米晶有很多优势：
•Qdot纳米晶激发范围宽，可被任何低于其发射峰的波长所激发。激发波长越低，消光系数和纳米晶亮度就越高。
•可以使用单一的激发波长进行Qdot纳米晶多色检测。
•Qdot纳米晶呈现出较大的斯托克斯位移。
•Qdot纳米晶发射光谱带较窄
•相比传统的荧光染料，Qdot纳米晶光稳定性好。
Qdot纳米晶由四个基本层组成，从内核到外壳依次为：
1.晶核（CdSe或CdSeTe）：决定了Qdot纳米晶的颜色
2.无机层（ZnS）：用于提高Qdot纳米晶的亮度和稳定性
3.有机/聚合物涂层：提供水溶性和/或用于偶联的官能团
4.生物分子：共价结合到聚合物层，包括免疫球蛋白、链霉亲和素、受体配体或寡核苷酸。
Here are our recommendations:
Use one of our extensive selection of secondary antibodies conjugated to bright, photostable Alexa Fluor dyes. The degree of labeling for each conjugate is 2-8 fluorophores per IgG molecule, with potentially three secondary antibody-binding sites per primary antibody, providing signal amplification of approximately 10-20 fluorophores per primary antibody.
Alternatively, primary antibody labeling can be detected with a biotinylated secondary antibody in conjunction with either a fluorescent streptavidin or a streptavidin bridge followed by a biotinylated reporter such as Qdot biotin. Although processing times increase with additional incubation and endogenous biotin-blocking steps, detection sensitivity also improves as a result of the labeled streptavidin.
For low-abundance targets, signal amplification may be necessary for optimal signal-to-noise ratios. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate reactive fluorophore-labeled tyramide radicals. These short-lived tyramide radicals covalently couple to nearby residues, producing an amplified fluorescent signal localized at the HRP-target interaction site.
For improved detection sensitivity with rapidly bleaching dyes, our SlowFade Diamond or ProLong Diamond antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
Please review this web page for further optimization tips (https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/guide-to-immunocytochemistry.html).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
A common method for amplifying antibody detection is biotin-streptavidin detection, where a biotinylated secondary antibody is combined with subsequent labeling with a dye-conjugated streptavidin. This will amplify the signal by approximately 2-8 times, but endogenous biotin must be blocked beforehand. Another option is to use tyramide-signal amplification, where a horseradish peroxidase conjugate is used with a dye-labeled tyramide. This will amplify the signal by approximately 10-20 times, but endogenous peroxidase will need to be blocked. A final option may be to use a Qdot nanoparticle antibody or streptavidin conjugate, which can yield a signal as much as 40 times higher than a standard organic dye conjugate, depending on the Qdot color.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Here are some suggestions:
Use the Qdot Incubation Buffer (Cat. No. Q20001MP).
The included buffer is formulated specifically for improved signal-to-background ratios in most immunolabeling applications using the Qdot streptavidin conjugates. Alternate buffers may result in more variable staining and, in particular, may increase background staining. However, some specific applications may require other buffer conditions. Please see the protocol "Double-labeling Using Qdot Streptavidin conjugates."
Determine if the sample has a high level of endogenous biotin. Block the sample using an avidin-biotin pre-blocking step.
If you have used the Qdot Incubation Buffer and still get high nonspecific background, then it may be necessary to check other steps of your procedure. Blocking the sample with BSA or normal animal serum will generally decrease nonspecific binding of both antibodies and Qdot streptavidin conjugates. It is a good practice to dilute your primary and secondary antibodies in the blocking buffer. Some tissues such as spleen and kidney sections may contain endogenous biotin, which may contribute to non-specific signal. Endogenous biotin can be blocked with an avidin/biotin blocking kit (Cat. No. E21390).
Grainy staining or clumps of fluorescent material appear in the background.
Occasionally the BSA within the Qdot Incubation Buffer shows slight aggregation over time. It is necessary to remove this aggregate prior to labeling the sample with the Qdot streptavidin conjugate. Spin down the incubation mixture before addition to the sample. This can be accomplished by spinning the samples in a benchtop centrifuge (Eppendorf 5415) at 5,000 x g for 2 minutes. The material can also be passed over a 0.2 µm spin filter unit before you add it to the sample for staining to remove microscopic precipitates. If you are using a buffer that is different than the Qdot Incubation Buffer, this behavior can often be attributed to higher levels of NaCl or other salts in the incubation buffer, and may not be easily fixed with filtration. In this case, reduce the overall salt concentration.
Optimize concentration of biotinylated secondary antibodies.
Optimizing specific signal can often be achieved by adjusting the level of biotinylated antibody used instaining. High levels of biotinylated antibody are necessary to obtain specific labeling, but overly high levels will contribute to nonspecific binding of the antibody to the sample. Nonspecifically bound biotinylated antibody will bind to the Qdot streptavidin conjugate, resulting in higher staining of the background.
Optimize concentration of Qdot streptavidin conjugate.
Just as titration of primary and secondary antibodies is necessary to achieve optimal specific signal in immunolabeling applications, the level of the final probe should be optimized for each conjugate. In general, concentrations at or slightly below saturation should have the optimal signal-to-background ratio, while concentrations substantially higher than saturation will compromise the assay with higher background levels.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Here are some suggestions:
Confirm imaging/detection setup suitability.
Make sure that you are using an appropriate filter set to detect the signal. Please consult Table 1 in the Qdot Biotin User Manual for a list of appropriate and optimal filters.
Check to see that Qdot conjugate is fluorescing using an alternative light source.
Qdot conjugates will normally fluoresce brightly under a hand-held ultraviolet lamp (long wave, such as the type used to visualize ethidium bromide on agarose gels). Although we have not seen pronounced loss of fluorescence of these materials under any storage conditions that we have investigated, we have not been able to examine all storage conditions. If the Qdot product does not appear to fluoresce under the long wave UV excitation, please contact Technical Support at techsupport@qdots.com. For a microscope, perform a spot test: place a small droplet (2 to 5 µL) of the quantum dot solution onto a clean slide (no coverslip) and examine under the appropriate filter set at low magnification.
Confirm the specificity and titer of primary antibody.
Make sure the antibody will recognize the intended targets. Make sure there is sufficient primary antibody bound to the targets. This verification can be performed by ELISA-based capture of the antigen of interest, or by other techniques that can be found in lab manuals such as the Current Protocols in Immunology.
For Qdot streptavidin conjugates, confirm biotinylation of antibody.
Make sure your antibodies are effectively biotinylated. It may be necessary to independently adjust the concentration of both the primary and secondary antibodies used in the assay to obtain optimal signal and minimal background.
PAP pen ink may quench signal.
Use an alternate method for isolating target areas on the slide. If your protocol requires the use of a PAP pen, we recommend the ImmEdge Hydrophobic Barrier Pen (Cat. No. H-4000) from Vector Labs.
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Spinning your ITK Qdot nanocrystals at approximately 3,000 rpm for 3-5 minutes should remove the white precipitate from the supernatant. Use the supernatant immediately.
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The precipitate in the organic ITK Qdot nanocrystals occurs with some frequency. The ITK Qdot nanocrystals sometimes include impurities that show as a white precipitate.
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Blinking is an inherent property of quantum dots; in fact, all single-luminescent molecules blink, including organic dyes. The brightness and photostability of Qdot nanocrystals makes the blinking more visibly apparent. Under higher energy excitation, Qdot nanocrystals blink even faster.
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Appropriate mounting media selection is very important to retain the fluorescence of Qdot nanocrystals. In our studies, Qdot nanocrystals work best with the following mountants:
HistoMount medium (Cat No. 00-8030); best for long term archiving
Cytoseal 60 Mountant
Clarion Mountant
Most polyvinyl alcohol-based mountants (limited storage time, less than weeks)
Water-based mountants (limited storage time, less than week)
Up to 50% glycerol (limited storage time, less than week)
Note: We do not recommend using ProLong mounting media with Qdot nanocrystals as it will quench their fluorescence.
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Freezing will cause the product to aggregate. The Qdot nanocrystals cannot be dispersed into solution after aggregation.
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Once your product undergoes aggregation, it cannot be dispersed back into solution. We recommend purchasing a new product.
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You may occasionally observe a small amount of aggregation of the Qdot nanocrystals during proper storage. To remove any aggregates that may have formed prior to use, we recommend centrifuging the vial at 2,000 x g for 1 min. Pipette only the supernatant and avoid the pellet. In our experience, pelleting any aggregates that may have formed typically results in a loss of less than 10% of the product.
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We have not systematically investigated the energy transfer properties of the quantum dots, though the quantum dots may have useful properties as both energy transfer donors and acceptors. We have investigated the fluorescence of Qdot 605 Streptavidin conjugates that are coupled to each other through a bis-biotin linker, and found that the emission intensity of the materials was unperturbed at any concentration of biotin cross-linker. These results suggest that the interparticle quenching of these Qdot conjugates is negligible.
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The Qdot products contain cadmium and selenium (and tellurium, in the larger particles) in an inorganic crystalline form. We can only advise that you dispose of the material in compliance with all applicable local, state, and federal regulations for disposal of these classes of material. For more information on the composition of these materials, consult the Material Safety Data Sheet.
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We have not investigated the toxicity of the Qdot nanocrystals. The materials are provided in a solution which is approximately 2 mM total Cd concentration. We have demonstrated the utility of these materials in a variety of live-cell in vitro labeling experiments, but do not have systematic data investigating the toxicity of the materials to humans, to animals, or to cells in culture.
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Qdot conjugates have higher nonspecific binding in buffers that are not optimized for use with the materials. We have had successful staining results in a variety of buffer conditions, including TBS, PBS, RPMI media, and others, but have found that the performance in the Incubation Buffer is generally predictable and stable.
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The Qdot 605 Streptavidin conjugates have stable emission in a number of buffers, across a range of pH. At working concentrations, the quantum yield and colloidal dispersion of these materials have been found to be remarkably stable across pH 6-9 (not investigated outside this range) in Tris, HEPES, phosphate, and borate buffers. The Qdot 605 Streptavidin conjugate is stable and non-aggregated in buffered NaCl up to 200 mM at working concentrations. Higher salt concentrations may result in microscopic precipitation, but do not appear to cause bulk precipitation of the materials at working dilutions. In addition, a number of surfactants and additives such as Tween 20, Triton X-100, Pluronic F-68, NDSB-201, and EDTA, among others have been shown to maintain the fluorescence in 0.05% concentrations. In contrast, gelatin and dextran sulfate were both found to promote aggregation of the Qdot 605 Streptavidin conjugate at 0.05% concentrations, and should be avoided in labeling applications. In general, we recommend storage of the Qdot nanoparticle conjugate at the concentration at which it was shipped, rather than at a higher dilution. Storing materials at working dilution over longer periods of time may result in substantial performance loss. While we have not characterized the stability of the other Qdot conjugates in this variety of buffers, we anticipate similar levels of stability.
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The number of molecules conjugated to one Qdot nanocrystal is based on the ratio of quantum dot:molecule used in the conjugation, the number of available binding sites on the Qdot nanocrystal, and the size of both the Qdot nanocrystal and the molecule of interest. In general, there are 2-3 antibodies, 4-5 biotin molecules, and 6-8 streptavidin molecules per Qdot nanocrystal.
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ITK Qdot nanocrystals use the original formulation of outer polymer provided in the first generation of the Qdot products; except for the Amine-PEG products, the outer polymer does not include PEG. The outer polymer of the standard Qdot nanocrystals includes PEG.
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There are approximately 80-100 functional groups of each Qdot ITK nanocrystal. We use a type of immunosorbent assay to determine the EC50 of each conjugate.
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Yes, you can visualize Qdot nanocrystals using a standard filter; they will excite at any wavelength below their emission. Keep in mind that the lower the excitation value the brighter the Qdot nanocrystal fluorescence output.
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Qdot nanocrystals do not require the use of antifades as they do not photobleach or fade in the same manner as a chemical dye. In our studies, Qdot nanocrystals work best with the following mountants:
- HistoMount medium (Cat No. 00-8030); best for long-term archiving
- Cytoseal 60 Mountant
- Clarion Mountant
- Most polyvinyl alcohol-based mountants (limited storage time, less than a week)
- Water-based mountants (limited storage time, less than a week)
- Up to 50% glycerol (limited storage time, less than a week)
Note: We do not recommend using ProLong or SlowFade mounting media with Qdot nanocrystals.
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Hydrophilic Qdot nanocrystals are stored and shipped in borate buffer pH 8.3-9.0, and organic Qdot nanocrystals are stored and shipped in decane.
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When stored at 4 degrees C, Qdot nanocrystals are stable for approximately 6 months. Qdot nanocrystals should never be frozen due to the possibility of aggregation. The temperature stability of Qdot nanocrystals is summarized below. Please note that fluorescence is not temperature dependent.
<0 degrees C: NEVER freeze Qdot nanocrystals - polymer induces aggregation at freezing temperatures.
>4 degrees C: Core/Shell/Polymer stable at 4 degrees C for ~ 6 months. May be filter sterilized using uncharged filters.
<60 degrees C: Core/Shell/Polymer stable at 60 degrees C (as in in situ hybridization).
<65 degrees C: Core/Shell/Polymer stable at 65 degrees C for only ~1 hour, beyond 1 hour, emission drops off.
<100 degrees C: Core/Shell/Polymer stable up to 100 degrees C brief exposure. OK for 5 minutes at 100 degrees C.
<360 degrees C: Only Core/Shell stable up to 360 degrees C.
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Qdot nanocrystals are most stable at pH 6-9, and marginal stability of Qdot nanocrystals is shown down to a pH 5. Qdot nanocrystals should not be used at pH > 9 due to the possibility of self-aggregation and clumping, and Qdot nanocrystals should not be used pH less than 4 as the polymer and exposed core/shell will begin to dissociate. For more information on Qdot nanocrystals and recommended pH ranges, see pH Ranges for Qdot Nanocrystals (https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/qdot-reg--nanocrystal0.html)
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You can use Qdot nanocrystals with FRET applications in two scenarios:
- Qdot nanocrystals as donors with fluorescent dyes as acceptors
- Lanthanide (terbium, europium, etc.) as donors with Qdot nanocrystals as acceptors
Note: You cannot perform FRET experiments using Qdot nanocrystals as both donor and acceptor.
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We offer amino (PEG), carboxyl, and streptavidin-functionalized Qdot Innovator's Tool Kit ITK Nanocrystals for the preparation of custom conjugates of proteins or other biomolecules. Amino (PEG)-derivitized forms can be coupled to isothiocyanates and succinimidyl esters or with native carboxylic acids using water-soluble carbodiimides. Carboxyl-derivitized forms can be coupled to amine groups of proteins and modified oligonucleotides. Streptavidin-derivitized forms can be bound with biotinylated conjugates to form stable labeled complexes.
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Qdot nanocrystals and bioconjugates are ideal for experiments requiring long-term photostability or single-excitation, multicolor analysis. Some example applications include:
- Flow cytometry
- Cell and tissue staining
- Cell tracking
- WesternDot western blotting
- In vivo imaging
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Qdot nanocrystals offer many advantages over traditional fluorescent dyes:
- Qdot nanocrystals have a broad excitation range, and they can be excited by any wavelength below their emission peak. The lower the excitation wavelength, the higher the extinction coefficient and Qdot nanocrystal brightness.
- Multicolor detection using Qdot nanocrystals can be done using a single excitation wavelength.
- Qdot nanocrystals exhibit a large Stokes shift.
- Qdot nanocrystals have a narrow emission band.
- Qdot nanocrystals have excellent photostability compared to traditional fluorescent dyes.
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A Qdot nanocrystal is comprises four basic layers. Listed from inner core to outer shell, these are:
1) Core nanocrystal (CdSe or CdSeTe): Determines the color of the Qdot nanocrystal
2) Inorganic shell (ZnS): Improves brightness and stability of the Qdot nanocrystal
3) Organic/polymer coating: Provides water solubility and/or functional groups for conjugation
4) Biomolecule: Covalently attached to the polymer shell and can include antibodies, streptavidin, receptor ligands, or oligonucleotides.
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