SYPRO™ Ruby Protein Gel Stain, 1 L - FAQs

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79 product FAQs found

我使用SYPRO Ruby蛋白质凝胶染色剂和其他蛋白质染色剂染色后，看到整个凝胶上出现横跨50–68 kDa区域的条带。这个条带是什么？如何能防止其形成？

您的样品或凝胶上样孔可能被皮肤或头发中的角蛋白污染。应在上样前用超纯水或电泳缓冲液冲洗凝胶孔。制备样品时，应穿戴实验服和手套；制备样品时，应使用保存于密封塑料袋中而非摆放在实验台上的微量离心管。

使用SYPRO Ruby蛋白质凝胶染色剂对凝胶染色后，一些预染分子量标记物显示为黑色条带。这是为什么？

蓝色染色剂会吸收红色波长的光，所以它们会吸收SYPRO Ruby染色剂的红色荧光。SYPRO Ruby染色剂也会与这些蛋白结合，但其信号会被有色染色剂淬灭，导致阴性染色并产生黑色条带。含蓝色蛋白的分子量标记物淬灭SYPRO Ruby荧光的实例是，BenchMark预染蛋白Ladder和SeeBlue Plus2预染标准品中的一些蛋白质。若溴酚蓝染色剂在电泳时未完全脱离凝胶，也会出现相同的现象，经SYPRO Ruby染色的凝胶随后使用Coomassie Blue染色剂染色时也会发生信号丢失。大部分其他有色染色剂不会淬灭SYPRO Ruby染色剂的信号，能够得到正常的蛋白染色条带。

使用SYPRO Ruby蛋白质凝胶染色剂染色后，凝胶上出现大量“斑点”。这是怎样形成的？如何能够去除或者防止其形成？

SYPRO Ruby蛋白质凝胶染色剂存放时间越久，凝胶上的斑点越多，这是因为SYPRO Ruby染色剂会随时间而发生自聚集。染色剂聚集在染色剂容器、溶液中的污染物或来自空气或手套的颗粒物（包括皮肤和空气中的角蛋白）周围，也会形成斑点。若使用SYPRO Ruby蛋白质凝胶染色剂孵育凝胶数小时或更长时间，染色剂会在容器边缘积累并随着容器的持续摆动而移动，特别是在脱色步骤中，从而形成斑点。灰尘、头发、手套粉末或掉落在凝胶或玻璃成像板上的衣服线头中的自发荧光颗粒物，在成像时也会形成非染色剂斑点。成像仪对凝胶表面特征的聚焦越好，形成的可见斑点越多。

为了将斑点形成和其他背景碎片数量降至最低，应保持实验室卫生整洁，使用电阻率大于18 megohm/cm的超纯水制备溶液，接触凝胶前用水冲洗掉手套上的粉末，使用无尘擦布并穿上实验服或者不要穿可产生大量线头的衣服，每次使用时都用乙醇冲洗染色容器并在下次使用前擦掉所有残留染色剂，每次放置凝胶前用乙醇和水冲洗和擦净玻璃成像表面。在染色和洗涤步骤之间，用乙醇擦掉染色托盘表面积累的染色剂。快速染色步骤应在90分钟内完成，这样可防止大部分斑点的形成。一旦斑点沉积在凝胶表面后，无法将其洗掉。

在凝胶3D图像中，斑点会呈现为较高的尖峰。这些斑点与蛋白质斑点或条带的3D图像看起来不同。一些图像分析软件包具有降噪算法，可轻松识别和去除这类像素化噪音。

SYPRO Ruby蛋白质凝胶染色剂已超过1年，并出现一些可见的沉淀物。能否通过过滤除去沉淀，减少染色凝胶表面的“斑点”？

SYPRO Ruby蛋白质凝胶染色剂保存超过一年会不稳定。染色剂溶液会逐渐析出沉淀（自聚集），对蛋白条带的染色力度降低，导致凝胶表面的“碎片”或“斑点”增多。由于染色剂会粘在大部分滤纸和滤膜上，因此无法通过过滤从染色剂溶液中除去沉淀。

我使用SYPRO Ruby对凝胶进行染色时，得到明亮的染色蛋白条带，而且在条带周围有黑色“阴影”。这是为什么？应如何解决？

条带周围的阴影表示由SDS造成了较高的染色背景。将凝胶放在10%甲醇/7%乙酸中脱色稍微久一点，约延长30分钟，然后用水彻底洗涤。为更好地去除凝胶上的SDS并降低起始背景染色，应在以后的实验中尝试延长凝胶固定时间，约增加30分钟。

我想在同一块凝胶中进行翻译后修饰的多重染色和总蛋白质染色（如SYPRO Ruby蛋白质凝胶染料）。我应该按什么顺序使用这些染料进行凝胶染色？

凝胶将需要被连续染色，并在每次染色后成像。染色顺序为：

InVision His-Tag In-Gel Stain（货号LC6030）→成像→ Pro-Q Diamond磷酸化蛋白凝胶染料→成像→ Pro-Q Emerald 300或488糖蛋白凝胶染料→ SYPRO Ruby蛋白质凝胶染料→ Coomassie Blue或银染→成像

经过Pro-Q Diamond磷酸化蛋白质印迹膜染料染色的印迹膜，能否再使用SYPRO Ruby印迹膜染料染色以进行总蛋白检测？

可以。我们推荐在使用Pro-Q Diamond磷酸化蛋白质印迹膜染料染色后，再使用SYPRO Ruby印迹膜染料染色。

SYPRO Ruby蛋白质印迹膜染料能否用于Immobilon CD或Immobilon N膜的染色？

不能。SYPRO Ruby蛋白质印迹膜染料不能用于阳离子膜，如Immobilon CD或Immobilon N膜。因为SYPRO Ruby蛋白质印迹膜染料是一种阴离子染料，它会与膜发生非特异性结合。酰胺黑、Coomassie Blue、丽春红和任何其他总蛋白印迹膜染料也是如此。建议将SYPRO Ruby蛋白质印迹膜染料用于Immobilon P或Immobilon FL膜。

在湿润还是干燥状态下，对SYPRO Ruby染色印迹膜进行成像更好？

SYPRO Ruby染料在干燥状态下更明亮，因此，干燥印迹膜更适用于照明成像。如果您只有透照仪，由于印迹膜在湿润时更透明，所以在湿润状态下成像会得到更明亮的信号和更低的背景。

能否对SYPRO Ruby染色印迹膜进行免疫染色？

可以。SYPRO Ruby染料可兼容后续的免疫检测，可使用比色、荧光和化学发光检测技术，包括BCIP/NBT、ECL和CDP-Star试剂。为获得最佳的免疫检测灵敏度，我们建议在免疫检测步骤前，使用含150 mM Tris的20%甲醇溶液（pH 8.8）进行脱色处理10分钟，随后用去离子水洗涤4次，每次1分钟，以去除大部分SYPRO Ruby染料。

干燥的SYPRO Ruby染色印迹膜可保存多久？

经过SYPRO Ruby印迹膜染料染色的干燥印迹膜的光稳定性很强，可长期保存在室温和避光条件下，包括夹在笔记本中。

为什么我们建议在转印后干燥PVDF膜，而不在固定和使用SYPRORuby印迹膜染料染色后用甲醇再润湿膜？有什么特殊原因吗？

是的，与用甲醇再润湿的印迹膜染色（浸没式染色）相比，对干燥的PVDF膜进行染色（面染色）可产生较低的非特异性背景信号和更好的信噪比。背景降低的另一个原因是，膜的背面没有染料。印迹膜上未完全干燥或有很多SDS残留的区域会湿透并呈现为较暗的染色背景。如果这是问题，那么可使用100%甲醇将整个PVDF印迹膜润湿，使整个背景的染色程度相同。染色蛋白质的信号和印迹膜的背景都会变得更明亮。您还应该增加水洗次数或延长印迹膜浸没染色的时间。干燥印迹膜也会使蛋白质完全结合到膜上。

对SYPRO Ruby染色印迹膜的清洗，能否超过硝化纤维素膜的推荐时间4–6次、每次1分钟或PVDF膜的推荐时间2–3次、每次1分钟？

可以。印迹膜可长期放置在水中，并且不会使染色的蛋白质脱色。长时间的清水洗涤有助于降低非特异性背景，特别是对于浸没式染色的PVDF膜。

我能否将印迹膜在SYPRO Ruby印迹膜染料中孵育超过15分钟？

可以，15分钟是最短时间。如果在SYPRO Ruby印迹膜染料中孵育超过15分钟，不会使印迹膜过度染色。

我能否从经过SYPRO Ruby染色的凝胶或印迹膜中完全去除染料？

不能完全去除。使用含150 mM Tris 的20–50%甲醇溶液（pH 9）孵育凝胶或PVDF膜并多次更换干净的溶液，可去除大部分染料，但不能完全去除。

经SYPRO Ruby、SYPRO Orange或SYPRO Red染色的凝胶能否干燥？

经过SYPRO染料染色的凝胶可放置在玻璃纸之间进行干燥，但有时会轻微降低灵敏度。如果在纸上干燥凝胶，光会散射且灵敏度会降低。不推荐使用其他塑料，因为通常使用的塑料是不可透过紫外光的。

SYPRO Ruby蛋白质凝胶染料对光漂白有多敏感？

使用Safe Imager 2.0蓝光透照仪时，需要8-10分钟持续紫外照射才能引起过度的光漂白。漂白率取决于光源强度，但在大多数情况下，这并不是问题。即使发生漂泊，也只会轻微降低SYPRO染料预染凝胶的灵敏度。

经SYPRO Ruby染色的凝胶保存多久，信号才会消失？

SYPRO Ruby凝胶染料的解离率非常低。实际上，染料与蛋白质结合后，很难将染料从凝胶上洗脱出来。染色凝胶可在4℃下避光保存至少几个月而无信号损失。将凝胶保存在含2–5 mM叠氮化钠（作为防腐剂）的少量水中或7%乙酸中，维持水化状态。对于更为长久的保存，可将凝胶干燥或真空密封保存在含2–5 mM 叠氮化钠的1–5 mL SYPRO Ruby染料中，并保存在4℃。可使用Seal-A-Meal食品保存袋。

将凝胶保存在SYPRO Ruby凝胶染料溶液中多久，不会导致过度染色？

SYPRO Ruby凝胶染料是一种终点染料，这表示一旦染料与蛋白质的结合达到饱和，则染料不会继续对蛋白质染色或增强背景信号，这一点与银染不同。凝胶可在SYPRO Ruby凝胶染料中保存几个月（建议在4℃）且不会过度染色。

我有一些SYPRO Ruby蛋白质凝胶染料/SYPRO Ruby蛋白质印迹膜染料。我能否使用凝胶染料对硝化纤维素或PVDF膜上的蛋白质染色，或使用印迹膜染料对凝胶染色？

不能，SYPRO Ruby蛋白质凝胶染料和印迹膜染料的配方有很大不同，它们经过优化分别适用于各自的用途，并且不可交换使用。交换使用印迹膜或凝胶染料进行染色，会产生不理想的染色强度和较高的背景。

我能否使用Coomassie Blue对经过SYPRO Ruby蛋白质染色的凝胶或印迹膜进行后染色？

可以，经SYPRO Ruby染色的凝胶和印迹膜可使用任何Coomassie Blue染料染色，产生的结果与只使用Coomassie Blue染料染色的凝胶和印迹膜相似。SYPRO Ruby的荧光将在Coomassie Blue染色后消失。

如果对经过SYPRO Ruby蛋白质染色的凝胶进行银染，会发生什么？

经SYPRO Ruby染色的凝胶可使用任何银染试剂进行后染色，如SilverXpress银染试剂盒（货号LC6100）和SilverQuest银染试剂盒（货号LC6070）。实际上，双重染色是增强单独SYPRO Ruby染色凝胶或银染凝胶灵敏度的极佳方法。只需在SYPRO Ruby染色后遵循常规的10%甲醇、7%乙酸脱色和清水洗涤步骤，如有需要可进行SYPRO Ruby信号成像，然后进行银染操作。银染实验中，无需固定步骤，因为凝胶已经被固定。银离子被大量吸引到SYPRO Ruby染料上，蛋白质周围的银沉积增加，从而增强了信号。当SYPRO Ruby染色凝胶经过银染后，SYPRO Ruby的荧光信号将不再被检测到。

SYPRO Ruby蛋白质凝胶染料可检测到的最小蛋白质是多大？

我们只测试了分子量低至6,000 Da的蛋白质，但是，如果有更小的蛋白质能够溶解在凝胶中并且含有赖氨酸、精氨酸或组氨酸碱性氨基酸残基，也有可能被检测到。

SYPRO Ruby蛋白质凝胶染料能否兼容质谱分析？

SYPRO Ruby染料不会影响质谱分析。使用SYPRO Ruby染料染色后，可对蛋白质条带或点进行胰酶消化，然后进行质谱分析。尽管SYPRO Ruby染料不会干扰质谱分析，但我们偶尔会看到一些由SYPRO Ruby染料形成的峰。对称的峰群集中分布在1257和1279 MW附近。

参考文献：Parker K, Garrels J, Hines W, Butler E, McKee A, Patterson D, Martin S. Electrophoresis 1998, 19, 1920–1932.

我需要剪切经SYPRO Ruby染色的凝胶条带/点。最好使用哪种灯箱？

在Safe Imager 2.0蓝光透照仪（货号G6600）或其他相似的蓝光透照仪中，SYPRO Ruby染料（以及SYPRO Orange、Coomassie Fluor Orange和核酸染料SYBR Safe、SYBR Gold以及SYBR Green I和II）在约470 nm激发波长处可很好地发出荧光。使用设备自带的琥珀色玻璃片，可观察凝胶。也可使用带有302 nm或365 nm灯泡的紫外灯透照仪，但使用时需要紫外防护设备。

我没有荧光成像仪，而且我只想对SYPRO Ruby染色的凝胶/印迹膜进行快速拍照。能否使用数码相机？

对于许多无需分析的常规凝胶或印迹成像，可使用简单的数码相机或拍照手机（如iPhone相机）并结合使用紫外或蓝光透照仪，如Safe Imager 2.0蓝光透照仪（货号G6600）。将凝胶置于透照仪表面，用琥珀色塑料滤镜覆盖凝胶。透过琥珀色塑料拍照，可降低背景亮度并提高灵敏性。对于印迹膜，最好从膜上方（照明）拍照，而非透过膜（透照）拍照。将灯箱放置在桌上，并将印迹膜面朝上放置在灯箱旁边。将琥珀色塑料滤镜拿到接近相机镜头的地方，透过滤镜拍照。

哪种仪器可用于SYPRO Ruby染色凝胶或印迹膜的成像？

点击这里（https://tools.thermofisher.com/content/sfs/manuals/x11791.pdf），查看用于SYPRO Ruby染料成像的仪器列表。

对于SYPRO Ruby凝胶染料使用手册第3页的快速染色方案，如果我没有足够的时间在同一天内完成实验，能否在某一点停止方案？

可以，最佳停止点是第一次固定步骤。凝胶可在第一次固定溶液中保存数天，并且对染色结果无影响。一次较长时间的固定足以去除凝胶上的SDS，因此无需重复固定步骤，这样可节约溶剂。过夜或更长时间的固定会使凝胶基质显著脱水，凝胶尺寸会减小并变成不透明的白色。微波处理前，必须再水化凝胶。在水中或SYPRO Ruby凝胶染料中摇动5分钟，可使凝胶再水化。在微波处理前，应确保凝胶漂浮在溶液中而没有粘到染色盘底部，否则，会发生不均匀的再水化或染色。当凝胶经过再水化而回到原始尺寸后，即可将其置于SYPRO Ruby凝胶染料中进行微波处理。

根据SYPRO Ruby蛋白质凝胶染料手册的步骤2.2，是否必须严格遵守微波染色实验方案中的23分钟孵育时间？

不是，23分钟恰好是30分钟总染色时间减去微波和5分钟孵育时间后剩下的时间。事实上，这些是所用的最短时间，从而使全部固定、染色、脱色步骤可在90分钟内完成。无需严格遵守微波染色步骤2.2的准确时间。凝胶染色时间可以长于30分钟，但背景也会随蛋白质信号逐渐增强，因此，微波方案的染色时间长于30分钟不会改善灵敏度。通常，染色30至90分钟会产生相似的结果。最高的终点信号强度可在5小时后达到，与过夜染色方案达到的结果相同。

在30分钟的染色时间内，只需将凝胶微波处理2次至将近沸腾，这样只会形成小气泡。过度沸腾会导致染色溶液撞击，在某些情况下，可使凝胶烧焦和损坏。

使用SYPRO Ruby凝胶染料染色前，一定要在50%甲醇/7%乙酸中固定凝胶吗？或者能否使用其他固定溶液？

不一定，也可使用其他常用的凝胶固定剂，包括10%甲醇/7%乙酸。SYPRO Ruby染料本身即可固定凝胶中的蛋白质，因此，对染色结果较好的蛋白质无需单独采用固定步骤。实验方案推荐使用的50%甲醇/7%乙酸固定剂，已被证明对于去除凝胶基质中残留的SDS效果最好，因此，与其他固定方法相比，该固定方法的背景最低、灵敏度最佳。降低甲醇浓度可能导致凝胶底部出现严重染色的SDS区域，从而降低在此区域附近电泳的低分子量蛋白质的检测灵敏度。

使用SYPRO Ruby凝胶染料染色前，能否使用乙醇/乙酸代替甲醇/乙酸固定凝胶？

可以，但是乙醇/乙酸会产生气味强烈的乙酸乙酯，因此，应在通风橱里进行固定。

我能否使用SYPRO Ruby、SYPRO Orange或SYPRO Red蛋白质凝胶染料对蛋白质进行预染，然后将蛋白质在凝胶上进行电泳？

不能。上样溶液含有很多SDS，因此，SYPRO Ruby、SYPRO Orange和SYPRO Red染料会只与游离的SDS结合，而极少与蛋白质结合。在电泳前，可预先使用ATTO-TAG CBQCA（货号A2333）、DDAO琥珀酰亚胺酯（货号C34553）或TAMRA-琥珀酰亚胺酯（货号C2211）染料或TC-FLAsH表达分析检测试剂盒（货号A10067为橙黄色荧光，货号A10068为红色荧光）对蛋白质进行共价标记，不会影响蛋白质在凝胶中的迁移。

电泳期间，可将SYPRO Orange或SYPRO Red蛋白质凝胶染料稀释5000倍并加入到阴极（上层）缓冲液槽中，对蛋白质进行染色而不影响迁移。这样做可能引起的问题是，染料与SDS的相互作用可能导致凝胶产生背景荧光。在电泳后使用7.5%乙酸脱色15–60分钟，可降低这种背景荧光。这种染色方法还会导致蛋白质灵敏度低于标准的后染色方法、在凝胶成像前需要相同的时间以及污染电泳装置。

经SYPRO Ruby蛋白质凝胶染料染色后，SDS-PAGE凝胶中的蛋白质能否转印到PVDF或硝化纤维素膜上？

对于使用SYPRO Ruby蛋白质凝胶染料或任何凝胶染料染色的蛋白质，若蛋白质固定在凝胶中，则不能将其转印到膜上。固定步骤和SYPRO Ruby凝胶染色溶液的低pH条件，都可将蛋白质沉淀到凝胶基质中，以防止蛋白质在染色过程中发生扩散，从而也阻止了蛋白质有效转印到膜上。我们推荐使用SYPRO Tangerine蛋白质凝胶染料，它是一种具有中性pH、以简单的盐溶液为基础的非固定型凝胶染料，可用于转印前的蛋白质预染。

SYPRO Ruby蛋白质凝胶染料能否稀释或重复利用？

SYPRO Ruby蛋白质凝胶染料可用水稀释最多5倍，这只会使检测灵敏度轻微降低。但是，即使是1/2稀释，也会使荧光信号和动态范围显著下降。重复利用染料多达2次，检测灵敏度依然很高，但是，染料在第二次使用时的信号强度会降低多达2.5倍。重复利用染料时，推荐将染料体积增加至100 mL。

参考文献：Ahnert N, Patton WF, Schulenberg B. Optimized conditions for diluting and reusing a fluorescent protein gel stain. Electrophoresis 2004, 25, 2506–2510.

哪种类型的蛋白质使用SYPRO Ruby蛋白质凝胶染料的染色效果不是很好？

如果一种蛋白质具有较低的碱性残基含量，则染色水平相对于高碱性残基含量的蛋白质较低。严重糖基化的蛋白质就是一种碱性残基含量较低的蛋白质，其染色效果可能不如非糖基化或轻度糖基化蛋白质的效果好，因此检测灵敏度较低。不含有任何碱性残基的蛋白质（可作为合成结构），不会与染料结合。

SYPRO Ruby蛋白质凝胶染料的线性动态范围是多少？

荧光强度随蛋白质含量呈线性变化，跨越将近3个数量级，从1 ng至1000 ng。SYPRO Ruby染料比银染的线性范围更宽。基本上，蛋白质越多，SYPRO Ruby染料的结合量越高。

SYPRO Ruby蛋白质凝胶染料与其他总蛋白质染料相比，效果如何？

由于SYPRO Ruby蛋白质凝胶染料主要与碱性蛋白质残基结合，而非SDS，因此不能将其作为终点染料（直接的饱和结合，并具有较低的解离率）。终点染料的性能不会受到固定、染色或脱色溶液体积或孵育时间的差异的影响，只受蛋白质含量的影响。SYPRO Ruby染色方案中较强的50%甲醇/7%乙酸固定，可除去大部分与蛋白质结合的SDS以及凝胶基质中的SDS，因此信号仅来源于直接的蛋白质结合。通过SDS/蛋白质结合作用而与蛋白质间接结合的染料（如Deep Purple、Lucy、SYPRO Orange/Red/Tangerine、Nile Red染料），会产生较大的蛋白质间以及凝胶间差异，这取决于与蛋白质结合的SDS量和凝胶基质中的SDS量。未与SDS饱和结合的蛋白质，如严重糖基化或磷酸化的蛋白质，使用间接SDS结合蛋白质染料的染色程度较低。

SYPRO Ruby蛋白质凝胶染料的亲和力是多少？

SYPRO Ruby蛋白质凝胶染料主要与蛋白质结合，通过染料的离子电荷结合到蛋白质的碱性侧链（赖氨酸、精氨酸、组氨酸以及较少的酪氨酸和色氨酸）上。固定液和SYPRO Ruby染色溶液均为酸性pH，SYPRO Ruby染料结合随质子化碱性残基的增加而增加。SYPRO Ruby染料也会结合与蛋白质结合的SDS以及凝胶基质中的SDS。固定溶液中较高的50%甲醇浓度，有助于去除凝胶基质中的SDS，从而降低背景染色并获得最佳的信噪比。

以下是一篇关于SYPRO Ruby蛋白质凝胶染料的氨基酸特异性的参考文献：
Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Steinberg TH, Chernokalskaya E, Berggren K, Lopez MF, Diwu Z, Haugland RP, Patton WF. Electrophoresis 2006, 21, 486–496.

对于经过SimplyBlue SafeStain染色的凝胶，我想使用SYPRO Ruby蛋白质凝胶染料再次染色。染色的最佳停止点是什么？

我们建议将凝胶放在染料中过夜。根据使用手册的建议，无需额外加入盐溶液，因为无需担心缓冲液盐和SDS，而且这样做会抑制多度染色。

Can proteins from 1D SDS-PAGE gels be transferred to PVDF or nitrocellulose after being stained with SYPRO Ruby protein gel stain?

No. Proteins stained with SYPRO Ruby protein gel stain cannot be blotted onto membranes. The fixation step and the fixative-like solution that the dye is dispersed in prevents efficient blotting of proteins onto membranes.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am getting a broad 50-68 kDa band across the entire length of the gel when I stain with SYPRO Ruby Protein Gel Stain and other protein stains. What is this and how can I prevent it?

Your samples or the gel wells were contaminated with keratins from skin or hair. Rinse out the gel wells with ultrapure water or running buffer before loading samples. Wear a lab coat and gloves when preparing samples and use microfuge tubes that have been stored in sealed plastic bags, not left out on the bench top, for preparing samples.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Some of my pre-stained molecular weight markers are appearing as dark bands when I stain the gel with SYPRO Ruby Protein Gel Stain. What is causing this?

Blue-colored dyes absorb light in the red wavelengths, so they absorb the red fluorescent emission of SYPRO Ruby dye. SYPRO Ruby dye still binds these proteins, but the signal is quenched by the colored dye, resulting in a negatively stained, dark band. Examples of molecular weight markers with blue-colored proteins that will quench SYPRO Ruby fluorescence are the BenchMark Pre-Stained Protein Ladder and some proteins in the SeeBlue Plus2 Pre-Stained Standard. The same phenomenon can be seen with the bromophenol blue dye front, if it is not completely run off the gel, and loss of signal when SYPRO Ruby stained gels are subsequently stained with Coomassie Blue stains. Most other colored dyes do not quench the SYPRO Ruby dye signal and will appear as normally stained protein bands.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My gels are showing a large amount of ‘speckles' after staining with SYPRO Ruby Protein Gel Stain. How are they formed and what can I do to remove them or prevent them from even forming?

Speckles on the gel can increase as the SYPRO Ruby Protein Gel Stain ages, due to self-aggregation of the SYPRO Ruby dye over time. Speckles can also form due to dye aggregation around contaminants from the staining container, solutions, or particles from the air or gloves, including keratin proteins from skin and hair. When gels are incubated with SYPRO Ruby Protein Gel Stain for several hours or longer, dye can build up on the sides of the staining container and then be dislodged with continuing rocking, especially during the destain step, forming speckles. Non-dye speckles can also show up in the image from auto-fluorescent particles of dust, hair, glove powder, or clothing lint that falls on the gel or surface of the glass imaging plate. The better the imager is at focusing on surface features of the gel, the more speckles that are going to be visible.

To minimize the formation of speckles and other background debris, follow clean laboratory practices, use ultrapure water of greater than 18 megohm-cm resistance to prepare solutions, rinse gloves in water to remove powder residue before touching gels, use lint-free wipes and wear a lab coat or avoid wearing clothing that generates a lot of lint, always rinse the staining container with ethanol and wipe out any residual dye before staining another gel, and always rinse and wipe down the glass imaging surface with ethanol and water before placing your gel down. Remove dye buildup on the surface of the staining dish by wiping out the dish with ethanol between the stain and wash step. The rapid stain protocol is complete in as little as 90 minutes, which does not allow enough time for most speckles for form. Once speckles have been deposited on the gel, it is not possible to wash them off. Speckles will show up as sharp, tall spikes on 3D renditions of gel images. These spikes look distinct from 3D renditions of protein spots or bands. Some image analysis software packages have de-speckling algorithms that can easily identify and remove this type of pixelated noise.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My SYPRO Ruby Protein Gel Stain is more than a year old and has some visible precipitate. Can I filter the stain to remove the precipitate and reduce the amount of ‘speckles' on the surface of my stained gels?

SYPRO Ruby Protein Gel Stain is not stable beyond about a year. The dye begins to precipitate out from solution (self-aggregate) over time and will show a lower staining intensity of protein bands and increased ‘debris' or ‘speckles' on the surface of the gel. It is not possible to filter the stain to remove dye precipitate, as the dye sticks to most paper and membrane filters and will be removed from the staining solution.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My SYPRO Ruby stained gel shows brightly stained protein bands with dark "shadows" around the protein bands. What causes this and what can I do to remedy it?

Shadowing around the bands means that the gel background staining of SDS is too high. Destain the gel in 10% methanol/7% acetic acid a little longer, approximately another 30 minutes and then give it a good water wash. In the future, try fixing the gel longer, at least another 30 minutes, to better remove SDS from the gel and reduce initial background staining.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I wish to multiplex stains for post-translational modifications and a total protein stain such as SYPRO Ruby Protein Gel Stain in the same gel. In what order should I stain the gel with these stains?

The gel would need to be sequentially stained and imaged after each stain. The order of staining would be: InVision His-Tag In-Gel Stain (Cat. No. LC6030) then Image then Pro-Q Diamond Phosphoprotein Gel Stain then Image then Pro-Q Emerald 300 or 488 Glycoprotein Gel Stain then SYPRO Ruby Protein Gel Stain then Coomassie Blue or Silver Stain then Image

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have stained my blot with Pro-Q Diamond Phosphoprotein Blot Stain. Can I also stain the blot with SYPRO Ruby Blot Stain for total protein detection?

Yes. We recommend staining with SYPRO Ruby Blot Stain after staining with Pro-Q Diamond Phosphoprotein Blot Stain.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I use Immobilon CD or Immobilon N membranes with SYPRO Ruby Protein Blot Stain?

No. SYPRO Ruby Protein Blot Stain is not compatible with cationic membranes, such as Immobilon CD or Immobilon N membranes. Since it is an anionic dye, it binds non-specifically to the membrane. This is also true for amido black, Coomassie Blue, ponceau red and just about any other total protein blot stain. The Immobilon P or Immobilon FL membranes are recommended for use with SYPRO Ruby Protein Blot Stain.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Is it better to image my SYPRO Ruby stained blot wet or dried?

SYPRO Ruby stain is actually brighter when dry, so a dry blot is better for epiillumination. If you only have a transilluminator, blots are more transparent when wet, so wet illumination will give a brighter signal and lower background.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I immunostain my SYPRO Ruby stained blot?

Yes. SYPRO Ruby stain is compatible with subsequent western detection using colorimetric, fluorogenic, and chemiluminescent detection techniques including BCIP/NBT, ECL, and CDP-Star reagent. For best western detection sensitivity, we recommend destaining the blot in 20% methanol, 150 mM Tris, pH 8.8 for 10 min followed by four 1 min rinses in deionized water to remove most of the SYPRO Ruby dye before performing the western detection procedure.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How long can I retain my dry SYPRO Ruby stained blot?

Dried blots stained with SYPRO Ruby blot stain are very photostable and can be stored for long periods of time at room temperature, in the dark, including taping into a notebook.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Is there a specific reason why we recommend drying PVDF membranes after transfer, and not re-wetting the membrane in methanol before fixation and staining with SYPRO Ruby Blot Stain?

Yes, staining a dry PVDF membrane (face staining) gives a lower non-specific background signal and results in a better signal to noise compared to a blot that has been re-wet in methanol (immersion staining). Background is also lower because the back side of the membrane does not see stain. Areas of the blot that are not completely dry or have a lot of residual SDS will wet out and show up as a darker stained background. If this is a problem, the entire PVDF blot can be re-wet in 100% methanol, so that the entire background is stained the same. Both the stained protein signal and blot background will be brighter. You should also increase the number of water washes or time for immersion stained blots. Drying the blot also completely binds the proteins to the membrane.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I wash my SYPRO Ruby stained blots longer than the recommended 4-6 times/1 min in deionized water for nitrocellulose membranes or 2-3 times/1 min for PVDF membranes?

Yes. Blots can be left in water indefinitely without destaining the stained proteins. Longer water washes can help to reduce high non-specific background, especially for immersion-stained PVDF membranes.

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Can I incubate my blot in SYPRO Ruby Blot Stain longer than 15 min?

Yes, the 15 min incubation time is a minimum time. The blot will not overstain if left in SYPRO Ruby Blot Stain longer than 15 min.

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Can I completely remove the dye from my SYPRO Ruby stained gel or blot?

No, not completely. You can incubate the gel or PVDF blot in several changes of 20-50% methanol, 150 mM Tris, pH 9 to remove the majority of the dye, but it will not be completely gone.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I dry my SYPRO Ruby, SYPRO Orange, or SYPRO Red stained gels?

Gels stained with SYPRO dyes can be dried between sheets of cellophane, although there is sometimes a slight decrease in sensitivity. If the gels are dried onto paper, the light will scatter and the sensitivity will decrease. Other plastics are not recommended, as the plastic typically used is not transparent to UV light.

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How sensitive is SYPRO Ruby Protein Gel Stain to photobleaching?

With the Safe Imager 2.0 Blue-Light Transilluminator, it takes 8-10 minutes of constant ultraviolet illumination to cause excessive photobleaching. The bleaching rate will vary with the intensity of your light source, but under most conditions this probably will not be a problem. Even when bleached, gels can be prestained with SYPRO stains with only a small decrease in sensitivity.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How long can I keep my SYPRO Ruby stained gel before I can no longer see a signal?

SYPRO Ruby Gel Stain has a very slow off-rate. In fact, it is very difficult to completely strip the dye out of the gel once it has bound proteins. Stained gels can be stored at 4 degrees C, protected from light, for at least several months with little loss in signal. Just keep hydrated in a little water with 2-5 mM sodium azide as a preservative or in 7% acetic acid. For more permanent storage, dry the gel or vacuum seal in 1-5 mL of SYPRO Ruby stain containing 2-5 mM sodium azide and store at 4 degrees C. Seal-A-Meal food storage bags are useful for this method of preservation.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How long can I keep my gel in SYPRO Ruby Gel Stain solution without over-staining the gel?

SYPRO Ruby Gel Stain is an endpoint stain, which means that once it has reached saturation binding of proteins, it will no longer continue to stain proteins or increase background signal, unlike silver stains. Gels can be left in SYPRO Ruby Gel Stain for months (4 degrees C recommended) without over-staining.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have some SYPRO Ruby Protein Gel Stain/SYPRO Ruby Protein Blot stain. Can I use the gel stain to stain proteins on nitrocellulose or PVDF membranes or use the blot stain to stain my gel?

No, the SYPRO Ruby Protein Gel Stain and Blot Stain have very different formulations that are optimized for their respective usage and are not interchangeable. Blots or gels stained with the alternate SYPRO Ruby product will have suboptimal staining intensities and high backgrounds.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I post-stain my SYPRO Ruby protein-stained gel or blot with Coomassie Blue?

Yes, SYPRO Ruby stained gels and blots can be stained with any Coomassie Blue dye-based stain and will yield similar results as a gel or blot stained only with Coomassie Blue dye. The fluorescence of SYPRO Ruby will be lost after Coomassie Blue staining.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What would happen if I silver stained my SYPRO Ruby protein-stained gel?

SYPRO Ruby stained gels can be post-stained with any silver stain, such as SilverXpress Silver Staining Kit (Cat. No. LC6100) and SilverQuest Silver Staining Kit (Cat. No. LC6070), and double staining is actually an excellent method to enhance the sensitivity obtained with individually stained SYPRO Ruby or silver stained gels. Simply follow the normal 10% methanol, 7% acetic acid destain after SYPRO Ruby staining, wash in water, if desired, to image the SYPRO Ruby signal and then perform the silver stain procedure. It is not necessary to perform the fixation step in the silver stain protocol, as the gels have already been fixed. The silver ions are apparently attracted to the SYPRO Ruby dye, enhancing the silver deposition around the proteins and thus the signal. Once a SYPRO Ruby stained gel has been silver stained, the SYPRO Ruby fluorescence signal can no longer be detected.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How small of a protein can be detected with SYPRO Ruby Protein Gel Stain?

We have only tested proteins as small as 6,000 Da, but if smaller proteins can be resolved in the gel and contain lysine, arginine or histidine basic amino acid residues, then it is likely that they will be detected too.

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Is SYPRO Ruby Protein Gel Stain compatible with mass spectrometry?

The SYPRO Ruby stain does not interfere with mass spectrometry. After staining with SYPRO Ruby stain, the protein band or spot can be trypsinized and sent for mass spectrometry analysis. Although the SYPRO Ruby stain does not interfere with mass spectrometry, occasionally we see some peaks that are due to the SYPRO Ruby dye. These are a symmetrical grouping of peaks centering around 1257 and 1279 MW.

Reference: Parker K, Garrels J, Hines W, Butler E, McKee A, Patterson D, Martin S. Electrophoresis 1998, 19, 1920-1932.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I need to cut out my SYPRO Ruby stained gel bands/spots. What type of light box would be best to use?

SYPRO Ruby dye (as well as SYPRO Orange, Coomassie Fluor Orange and the nucleic acid stains SYBR Safe, SYBR Gold, and SYBR Green I and II) fluoresces nicely on the Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600) or other similar blue light transilluminators with excitation near 470 nm. The gel can be viewed with amber glasses that are supplied with the unit. UV light transilluminators equipped with 302 nm or 365 nm bulbs can be used as well, but would require UV-protective equipment during use.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I do not have a fluorescence imager and just want to take a quick picture of my SYPRO Ruby stained gel/blot. Can I use my digital camera?

For many routine gel or blot images that do not require analysis, a simple digital camera or camera phone, such as an iphone camera, can be used in combination with a UV or blue light transilluminator, such as the Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600). Place the gel on the surface of the transilluminator and cover with the amber plastic filter. Take a picture through the amber plastic to reduce background illumination and improve sensitivity. Blots are best imaged from above the membrane (epiillumination) rather than through the membrane (transillumination). Place the light box on its side with the blot face-up on the table. Hold the amber plastic filter up close to the camera lens to take a picture.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Which instruments can I use to image my SYPRO Ruby stained gel or blot?

A handy list of instruments that work well with SYPRO Ruby stain is available here (https://tools.thermofisher.com/content/sfs/manuals/x11791.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

For the SYPRO Ruby Gel Stain Rapid Protocol on Page 3 of the manual, is there any point where I can stop the protocol, if I do not have time to finish in the same day?

Yes, the best place to stop for the day is during the first fixation step. Gels can be left overnight up to several days in the first fixation solution with no effect on the resulting staining. One long fixation is sufficient to remove SDS from the gel, so it is not necessary to repeat the fixation step, thus reducing solvent usage. An overnight or longer fixation will dehydrate the gel matrix significantly, so that it will be reduced in size and turn opaque white. You must rehydrate the gel before microwaving. Simply rock for about 5 min in water or SYPRO Ruby Gel Stain to rehydrate the gel. Check to make sure that the gel is floating in solution and has not stuck to the bottom of the staining dish before microwaving, or it will rehydrate unevenly and stain unevenly. After the gel has rehydrated back to its original size, it can then be microwaved in the SYPRO Ruby Gel Stain.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Per step 2.2 of the SYPRO Ruby Protein Gel Stain manual, must the 23 min incubation time for the microwave staining protocol be followed stringently?

No, 23 min happens to be the time that is left over from the total 30 min stain time after subtracting the microwave and 5 min incubation times. The reality is that those are the minimum times used so that the whole fix, stain, destain procedure can be complete in 90 min. The exact timing of the microwave staining step 2.2 does not need to be followed stringently. Gels can be stained longer than 30 min, but background will also gradually increase along with the protein signal, so that sensitivity is not improved by staining longer than 30 min using the microwave protocol. Generally, staining for 30 to 90 min will give similar results. The maximum end-point signal intensity is reached after about 5 h, the same as that achieved using the overnight staining protocol.

Gels just need to be microwaved twice during the 30 min stain time to near boiling, so that only small bubbles are formed. Over-boiling can bump the staining solution, and in some cases, scorch and damage the gel.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do I have to fix my gel in 50% methanol/7% acetic acid, or can other fixative solutions be used prior to staining with SYPRO Ruby Gel Stain?

No, other commonly used gel fixatives can be used, including reducing the methanol concentration to 10% methanol in 7% acetic acid. SYPRO Ruby stain itself will fix proteins in the gel and there is no need for a separate fixation step to stain proteins with reasonably good results. The 50% methanol/7% acetic acid fixation recommended in the protocol has been determined to best remove residual SDS from the gel matrix and thus give the lowest background and optimal sensitivity compared to other fixation methods. Reduced methanol concentrations could result in a heavily stained SDS front at the bottom of the gel, which will reduce detection sensitivity for low molecular weight proteins running near this region.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I fix my gels in ethanol/acetic acid rather than methanol/acetic acid prior to staining with SYPRO Ruby Gel Stain?

Yes, but ethanol/acetic acid will produce ethyl acetate, which has a strong odor, so you should do this fixation in a fume hood.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I pre-stain proteins with SYPRO Ruby, SYPRO Orange or SYPRO Red Protein Gel Stains and then run them through a gel?

No. Loading solutions contain so much SDS that SYPRO Ruby, SYPRO Orange and SYPRO Red dyes simply localize in the free SDS and bind very little of the proteins. Proteins can be covalently pre-labeled with ATTO-TAG CBQCA (Cat. No. A2333), DDAO succinimidyl ester (Cat. No. C34553) or TAMRA-succinimidyl ester (Cat. No. C2211) dyes, or the TC-FLAsH Expression Analysis Detection Kits (Cat. No. A10067 for orange fluorescence, Cat. No. A10068 for red fluorescence) prior to electrophoresis without affecting protein migration through the gel.

SYPRO Orange or SYPRO Red Protein Gel Stain can be diluted 5000-fold into the cathode (upper) buffer tank to stain proteins during electrophoresis without affecting migration. The problem with doing this is that there is considerable background fluorescence in the gels from the dye interacting with SDS. This can be reduced after electrophoresis by destaining in 7.5% acetic acid for 15-60 minutes. This method also results in poorer protein sensitivity than the standard post-staining method, requires the same amount of time before the gel can be imaged, and contaminates the electrophoresis apparatus.

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Can proteins from my SDS-PAGE gel be transferred to PVDF or nitrocellulose after being stained with SYPRO Ruby Protein Gel Stain?

Proteins stained with SYPRO Ruby Protein Gel Stain, or any gel stain where the proteins are fixed in the gel, cannot be blotted onto membranes. Both the fixation step and the low pH of the SYPRO Ruby Gel Stain solution precipitate proteins into the gel matrix to prevent diffusion during staining, and thus also efficient transfer onto membranes. We recommend using SYPRO Tangerine Protein Gel Stain, which is a neutral pH, simple saline solution-based, non-fixative gel stain to prestain proteins before transfer.

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Can I dilute or reuse SYPRO Ruby Protein Gel Stain?

Detection sensitivity is only slightly diminished by dilution of SYPRO Ruby Gel Stain in water up to 5-fold. However, both the fluorescence signal as well as the dynamic range are both reduced significantly with even a 1/2 dilution. Detection sensitivity also remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice-used stain. Increasing the staining volume to 100 mL is recommended when reusing the stain.
Reference: Ahnert N, Patton WF, Schulenberg B. Optimized conditions for diluting and reusing a fluorescent protein gel stain. Electrophoresis 2004, 25, 2506-2510.

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Are there types of proteins that do not stain well with SYPRO Ruby Protein Gel Stain?

If a protein has a low content of basic residues, you will see a lower level of staining relative to a protein with a higher content of basic residues. Very heavily glycosylated proteins are an example of proteins with low content of basic residues and may not stain as well as non-glycosylated or lightly glycosylated proteins, thus lowering detection sensitivity. If a protein does not have any basic residues, as can exist for synthetic constructs, it will not bind the dye.

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What is the linear dynamic range of SYPRO Ruby Protein Gel Stain?

Fluorescence intensity varies linearly with protein quantity over nearly three orders of magnitude, from 1 ng to 1000 ng. SYPRO Ruby stain has a broader linear range than silver staining, basically, the more protein you have, the more SYPRO Ruby stain that binds.

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How does SYPRO Ruby Protein Gel Stain compare to other total protein stains?

Since SYPRO Ruby Protein Gel Stain binds primarily to basic protein residues instead of SDS, it can be used as an endpoint stain (direct, saturation binding with a low off-rate) whose performance is not affected by deviations in fixation, staining or destaining solution volumes or incubation times, but just the amount of protein that is present. The strong 50% methanol/7% acetic acid fixation in the SYPRO Ruby stain protocol removes most SDS bound to proteins and in the gel matrix, so that the signal is due to direct protein binding. Stains that bind protein indirectly via SDS/protein association (such as Deep Purple, Lucy, SYPRO Orange/Red/Tangerine, Nile Red dyes) show higher protein to protein and gel to gel variation depending on how much SDS is bound to the protein and present in the gel matrix. Proteins that do not bind SDS to saturation, such as heavily glycosylated or phosphorylated proteins will show reduced staining with indirect SDS binding protein stains.

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What is the binding affinity for SYPRO Ruby Protein Gel Stain?

SYPRO Ruby Protein Gel Stain binds primarily to proteins through ionic charges of the dye, with basic side chains (lysine, arginine, histidine and to a lesser extent with tyrosine and tryptophan. The fixative solution and SYPRO Ruby stain solution both have an acidic pH, and SYPRO Ruby dye binding increases with protonated basic residues. SYPRO Ruby dye will also bind SDS bound to the proteins and in the gel matrix. The high 50% methanol concentration in the fixative solution is better at stripping out the SDS from the gel matrix, lowering the background staining and allowing for an optimal signal to noise.

Here is a reference on amino acid specificity of SYPRO Ruby Protein Gel Stain:
Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Steinberg TH, Chernokalskaya E, Berggren K, Lopez MF, Diwu Z, Haugland RP, Patton WF. Electrophoresis 2006, 21, 486-496.

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I would like to use SYPRO Ruby Protein Gel Stain to stain a gel that has been stained with SimplyBlue SafeStain. What is the best stopping point during staining?

We recommend leaving the gel in the stain overnight. There is no need to add the extra salt solution as recommended in the manual because there are no buffer salts and SDS to worry about, and that would end up inhibiting the staining too much.

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