UltraBio™ SDS-PAGE蛋白染色及上样缓冲液

S751584-1ml

1ml

5X

阿拉丁生产的UltraBio™ SDS-PAGE蛋白染色及上样缓冲液(5X)，即UltraBio™ SDS-PAGE Protein Staining and Loading Buffer (5X)，是一种添加了安全无毒的特殊荧光染料的SDS-PAGE蛋白上样缓冲液(5X)，能使蛋白在上样前与上样缓冲液的煮沸过程中被染色成紫色并带上橙黄色荧光，这样在电泳过程中或电泳后就可以直接肉眼观察到蛋白的紫色条带或者通过紫外或蓝光设备观察到蛋白的橙黄色荧光条带。本产品的原理和使用效果类似于VWR/Amresco的Protein EZ-Vision，都使用了安全无毒的荧光染料，无需额外的染色和脱色步骤，在蛋白和上样缓冲液煮沸的过程中就能完成染色步骤，在电泳完成后就可以直接观察和记录蛋白的电泳图谱，兼容紫外和蓝光检测系统，并且兼容后续的Western检测等。安全无毒，染色便捷：使用安全无毒的染料，并且没有任何额外的实验步骤，按照普通的SDS-PAGE蛋白上样缓冲液(5X)的操作步骤操作即可。蛋白样品加上1/4体积的本产品，混匀后，95-100℃水浴加热5分钟，就可以完成对蛋白样品的染色，把蛋白染色成紫色同时使蛋白带上橙黄色荧光。染色、电泳等可以在普通灯光下操作，无需采取额外的避光措施。不影响电泳迁移率：染料分子小并且不带电荷，不影响蛋白条带的电泳迁移速率和最终形成的电泳图谱。可以即时观察样品电泳效果：使用本产品在电泳结束后可即时观察蛋白样品的电泳效果。蛋白条带的量大于500ng时可直接肉眼观察到紫色蛋白条带，蛋白量较少时，可使用紫外灯或蓝光灯(需要配合使用蓝光滤光片)在暗室中观察蛋白条带的荧光。无需凝胶染色：使用本产品后无需进行任何凝胶染色，就可以直接拍照记录蛋白样品的电泳图了。蛋白量大时可以直接拍照，蛋白量比较少时，可以使用紫外或蓝光成像设备进行拍照和记录。兼容Western检测：使用本产品上样并电泳完成后，可以直接转膜并进行Western检测，无需任何额外的处理。PVDF膜和硝酸纤维素膜(NC膜)都能适用。转膜后可以同样观察蛋白的紫色条带或橙黄色荧光条带。通过对多种常用抗体的测试发现，使用本产品对于Western的检测灵敏度没有任何显著影响。国外同类产品也可以完美兼容Western检测。当然不能排除本产品可能会影响个别抗体对于目的蛋白的检测。背景极低，游离染料不影响观察和检测：游离的染料分子与示踪染料溴酚蓝有几乎相同的迁移速率，它们会共同电泳迁移至凝胶末端，确保凝胶的背景洁净透明。灵敏度高，低至25ng：使用本产品后，对于0.5μg或以上的单一蛋白条带，其经过荧光染料修饰后产生的粉红色略偏紫色的条带即使在日光灯下肉眼清晰可见(图1)，0.2μg的蛋白条带也隐约可见；使用紫外或蓝光成像设备，可以检测到低至25ng左右的蛋白条带(图2)。兼容紫外和蓝光成像系统：无论使用紫外成像设备，还是使用对人体无害的蓝光成像设备，都可检测低至25ng的蛋白条带。兼容Tris-glycine和Hepes凝胶体系：对于最常见的Tris-glycine凝胶体系和较常见的Hepes凝胶体系都能很好地兼容。染色稳定性好：SDS-PAGE电泳结束后，蛋白条带的荧光至少可以在4℃稳定保存5小时。如果电泳结束后立即转膜，膜上蛋白条带的荧光至少可以在4℃稳定保存24小时。主要 用途 ： Western检测时直接观察记录并确认每个泳道的上样量是否均衡，特别对于组织样品的Western检测尤为重要；SDS-PAGE时最快速便捷地观察到目的蛋白的诱导表达、分离纯化等效果。 注意事项 ： 低pH会显著影响染色效果。如果蛋白样品溶液的pH值低于5，为了获得更好的显色效果，建议将pH值调整至7以上。大多数预染和非预染的蛋白分子量标准与本产品不兼容。未添加上样缓冲液并且溶于水或生理盐水的非预染蛋白分子量标准，可以与InstantView? SDS-PAGE蛋白染色及上样缓冲液(5X)进行反应以制备成带有橙黄色荧光的蛋白分子量标准。经本产品进行染色的蛋白条带不推荐用于后续的蛋白测序、蛋白质谱分析或抗体制备。本产品须-20℃避光保存。室温避光放置时间过长(超过2-3周)，由于氧化会降低染色效果，此时可额外添加适量DTT，蛋白条带会重新变得清晰和明亮，但添加DTT的终浓度不要超过20mM。本产品对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)的配制。a.取出装有InstantView™ Protein Staining Dye的离心管，5000-10000g离心10-20秒以把染料离心至管底。b.取出InstantView™ Protein Loading Buffer (5X)，室温融解并混匀。低温时会产生SDS等形成的沉淀物，恢复至室温并混匀后沉淀会消失。c.在装有InstantView™ Protein Staining Dye的离心管中加入1ml InstantView™ Protein Loading Buffer (5X)，充分溶解并混匀后，就得到了InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)。d.配制好的InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)可以立即使用，或-20℃避光保存并在2个月内有效。2.InstantView™SDS-PAGE蛋白染色及上样缓冲液(5X)与蛋白样品按体积比1:4的比例混合。例如16μl蛋白样品加入4μl InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)。3.95-100℃水浴加热5分钟。如有必要，可将加热时间延长至10-15分钟，以确保细胞或组织样品中的基因组DNA断裂充分，避免出现粘稠状物。在个别染色效果欠佳的情况下，延长加热时间也可以确保蛋白充分变性并改善染色效果。4.当样品混合液温度降至室温时，即可按常规方法上样并进行SDS-PAGE凝胶电泳。5.如果需要适当的蛋白分子量标准，可以使用未添加上样缓冲液并且溶于水、生理盐水或PBS的非预染蛋白分子量标准。蛋白分子量标准的染色方法同普通的蛋白样品。6.电泳结束后，将凝胶转移至紫外或蓝光成像系统中进行观察和拍照。如果使用紫外光源须将凝胶从玻璃板中取出再进行观察和拍照；如果光源为蓝光，则无须把凝胶从玻璃板中取出。需要注意的是，在本产品使用过程中，正常的室内照明不会导致荧光的减弱，但如果在紫外灯下照射过长时间，会导致荧光逐渐减弱，即会出现荧光淬灭问题。7.如有必要，经InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)染色处理的凝胶仍然可以进行考马斯亮蓝染色。可以使用常规的考马斯亮蓝染色方法，例如阿拉丁的考马斯亮蓝染色试剂盒(常规法) ，但更推荐使用阿拉丁的考马斯亮蓝超快染色液，其中P0017F可在10分钟内检测到低至100ng的蛋白条带。8.后续如果进行Western检测，可以按照常规方法电转至PVDF膜或硝酸纤维素膜(NC膜)上，然后按照常规的Western检测方法进行Western检测。常见问题：1.InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)无法对蛋白分子量标准进行染色。大多数的预染和非预染蛋白分子量标准与InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)不兼容，请勿使用本产品处理这些蛋白分子量标准。2.InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)的稳定性。InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)较易被氧化，因此配制后的保存时间通常不宜超过一个月。室温条件下，不要超过1周。发现染色效果下降时，可以尝试加入不超过20mM的DTT或不超过2mM的TCEP作为还原剂，此时蛋白的染色效果通常会重新恢复正常。3.InstantView™ Protein Loading Buffer (5X)和InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)在低温条件下会有沉淀析出。InstantView™ Protein Loading Buffer (5X)和InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)中含有SDS，低温条件下会析出而形成沉淀，恢复至室温并混匀沉淀即会消失。4.InstantView™ Protein Staining Dye看不见。InstantView™ Protein Staining Dye的量非常少，确实肉眼很难分辨的。5.蛋白的翻译后修饰是否会影响InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)的染色效果？不会。6.InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)染色后的最佳激发波长？最佳激发波长是560nm，但在254nm、312nm、355nm、488nm和520nm都有很好的激发效果，这些激发光中总体上波长越长激发效果越好。7.使用InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)后是否还需要添加SDS-PAGE蛋白上样缓冲液(5X)？不需要，InstantView™ SDS-PAGE蛋白染色及上样缓冲液(5X)中已经包含了上样缓冲液。 aladdin‘’s UltraBio™ SDS-PAGE Protein Staining and Loading Buffer (5X) is used for loading protein samples onto SDS-PAGE gels for electrophoresis. It contains a safe and nontoxic fluorescent reagent that stains protein in purple during the boiling process and emits orange fluorescence under a UV or blue light. Post-run staining and destaining of SDS-GAGE gels are eliminated.This product is non-hazardous and convenient to use. Add 1/4 volume of this product to your protein sample, mix well and boil at 95-100ºC for 5 minutes. Proteins are then stained purple and emit orange fluorescence. All procedures can be performed under normal lighting without additional protection from light.The small and uncharged fluorescent molecules contained in UltraBio™ SDS-PAGE Protein Staining and Loading Buffer (5X) do not affect the migration rate of proteins and the resulting electrophoresis pattern.Electrophoresis results can be visualized immediately after the run. Proteins of more than 500ng can be directly observed by naked eyes. The fluorescence of proteins in small quantities can be observed in the darkroom using UV or blue light.This product is compatible with western blotting applications. Proteins in the SDS-PAGE gel can be transferred to PVDF or nitrocellulose membranes after electrophoresis. The purple band or orange fluorescence of proteins could also be observed on the membrane after transfer. The sensitivity of Western Blot detection is not affected by this product, which has been confirmed through testing a variety of commonly used antibodies. The possibility of affecting the detection of particular proteins can not be excluded.The background of gel after electrophoresis is low and free fluorescent dye does not affect observation and detection of proteins. The free fluorescent dye and the bromophenol blue tracking dye comigrate together to the end of the gel to ensure a clean background of the gel.This product has high sensitivity and can detect protein down to 25ng. A single protein band with more than 500ng can be observed obviously by naked eyes (Figure 1), and a 200ng protein band is faintly visible. Under a UV or blue light, proteins higher than 25ng can be observed (Figure 2).This product is compatible with the commonly used Tris-Glycine and Hepes gel buffer systems.The fluorescent signal is stable. The fluorescence of protein on SDS-PAGE gels is stable at 4ºC for at least 5 hours. If transferred immediately after electrophoresis, the fluorescence of protein on membranes is stable at 4ºC for at least 24 hours.Major Applications Application ： Rough comparison of protein quantity in each lane, especially for detection of tissue samples using Western Blot; Quick and convenient examination of protein induction, expression, and purification.Figure 1. Staining result of different amounts of BSA run with ’s InstantView™ Protein Staining and Loading Buffer (5X) . Mix different amounts of BSA proteins, as indicated in the figure, with an appropriate amount of this loading buffer, boil at 100ºC for 5 min, followed by electrophoresis. This photo was taken immediately after electrophoresis. The 500ng band can be observed obviously by naked eyes and the 200ng protein band is faintly visible.Figure 2. Images of protein samples run with ’s InstantView™ Protein Staining and Loading Buffer (5X) on SDS-PAGE gels. A and B. Mix different amounts of BSA proteins, as indicated in the figure, with the appropriate amount of this loading buffer, boil at 100ºC for 5 min, followed by electrophoresis. Picture A was taken immediately after electrophoresis under UV light and Picture B was taken immediately after electrophoresis under blue light. C. Stain result of bacteria cell lysate induced by IPTG to express a 35 kD target protein. Fluorescence was observed under UV light (left) after electrophoresis or under blue light (middle). The staining result with Coomassie Brilliant Blue is shown on the right.Packing List：ItemComponentQuantityP0280-1InstantView™ Protein Staining DyeFor 1mlP0280-2InstantView™ Protein Loading Buffer (5X)1mlManual-1 copyStorage Conditions：Store at -20℃ for up to 1 year. Store at 4℃ for up to 3 months. Store at room temperature for up to 2 weeks. The InstantView™ Protein Staining Dye is light-sensitive and should be protected from light.Precautions：Lower pH significantly affects protein staining by the InstantView™ SDS-PAGE Protein Staining and Loading Buffer. When the pH of protein samples is below 5, we recommend adjusting the pH to above 7.Most prestained and non-prestained protein molecular weight standards are incompatible with this product. Non-prestained protein molecular weight standards without any loading buffers or dissolved in water or saline can be mixed with an appropriate amount of InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) to produce protein molecular weight standards that emit orange fluorescence.Proteins stained by this product are not recommended for subsequent protein sequencing, protein profiling, or antibody preparation.InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) should be kept at -20℃ in the dark. Its staining performance reduces after 2-3 weeks at room temperature because of oxidation, which can be avoided by adding DTT in the loading buffer at a concentration of no more than 20mM.This product is hazardous. Please handle with care to avoid inhalation or direct contact with the human body.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Precautions ： Lower pH significantly affects protein staining by the InstantView™ SDS-PAGE Protein Staining and Loading Buffer. When the pH of protein samples is below 5, we recommend adjusting the pH to above 7.Most prestained and non-prestained protein molecular weight standards are incompatible with this product. Non-prestained protein molecular weight standards without any loading buffers or dissolved in water or saline can be mixed with an appropriate amount of InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) to produce protein molecular weight standards that emit orange fluorescence.Proteins stained by this product are not recommended for subsequent protein sequencing, protein profiling, or antibody preparation.InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) should be kept at -20℃ in the dark. Its staining performance reduces after 2-3 weeks at room temperature because of oxidation, which can be avoided by adding DTT in the loading buffer at a concentration of no more than 20mM.This product is hazardous. Please handle with care to avoid inhalation or direct contact with the human body.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1. Prepare the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X).a. Centrifuge the InstantView™ Protein Staining Dye to the bottom of the tube at 5,000-10,000g for 10-20 seconds.b. Thaw the InstantView™ Protein Loading Buffer (5X) at room temperature. SDS contained in the loading buffer precipitates when stored frozen, but will be resolubilized at room temperature.c. Add 1ml of InstantView™ Protein Loading Buffer (5X) into the InstantView™ Protein Staining Dye, mix and dissolve the dye thoroughly to generate the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X).Note: The prepared InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) should be used immediately or can be kept at -20℃ in the dark for up to 2 months.2. Mix the Protein Staining and Loading Buffer (5X) and protein samples at a ratio of 1:4. For example, add 4µl of Protein Staining and Loading Buffer (5X) into 16µl of protein sample. 3. Boil the mixture at 95-100ºC for 5 minutes, which can be extended to 10-15 minutes when necessary to ensure that the genome DNA from tissue or cell samples is broken completely to avoid the appearance of sticky materials. Prolonged heating time can also ensure adequate denaturation of proteins and improve staining results in cases where staining results are not satisfactory.4. Load samples onto SDS-PAGE wells after cooling to room temperature.5. If protein molecular weight standards are needed, non-prestained protein molecular weight standards without any loading buffers or dissolved in water, saline or PBS can be mixed with an appropriate amount of the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) to produce protein molecular weight standards after boiling. 6. After electrophoresis, place the gel absent of glass plates on a UV transilluminator to immediately visualize bands, but if using blue light for the observation, removal of glass plates is not necessary. Note 1: Fluorescent protein signal will decrease gradually as exposure time to UV is increased.Note 2: Proteins in gels can also be stained with Coomassie Brilliant Blue, such as Coomassie Blue Staining Solution ( InstantBlue Coomassie Staining Solution ( SDS-PAGE Protein Staining and Loading Buffer (5X).Most prestained and non-prestained protein molecular weight standards are not compatible with the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X). 2. Stability of the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X).The InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) is easily oxidized and should be used within one month after preparation. When stored at room temperature, it should be used within one week. When the staining performance decreased, DTT of no more than 20mM or TCEP of no more than 2mM as reducing reagent usually can recover its staining performance. 3. There are precipitates in the InstantView™ Protein Loading Buffer (5X) and the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) when stored in cold.The InstantView™ Protein Loading Buffer (5X) and the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) contain SDS that precipitates when stored frozen, but will be resolubilized at room temperature.4. InstantView™ Protein Staining Dye is invisible.The amount of InstantView™ Protein Staining Dye is indeed so little that it could not be seen by naked eyes.5. Do the post-translational modifications of protein affect its staining with InstantView™ Protein Staining and Loading Buffer (5X)?No, the post-translational modifications do not have any effect on protein staining. 6. Which wavelength is optimal for the excitation of protein bands run with the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X)?The optimal wavelength of excitation light is 560nm; however, the following wavelengths can be used: 254, 312, 335, 488 and 520nm. In general, the longer the wavelength of these excitation lights, the better the excitation.7. Is SDS-PAGE Loading Buffer (5X) still necessary for protein samples that have been mixed with the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X)?No, the InstantView™ SDS-PAGE Protein Staining and Loading Buffer (5X) acts as both the loading dye and the protein visualization dye.