10% SDS-PAGE一步法下层胶预混液

S743047-250ml

250ml

阿拉丁生产的6%、8%、10%、12%和15% SDS-PAGE一步法下层胶预混液，即SDS-PAGE One-step Resolving Gel Master Mix，提供了简单而又超快速地配制相应浓度SDS-PAGE凝胶(即SDS聚丙烯酰胺凝胶)下层胶所需主要试剂的混合溶液，该混合溶液中包含Tris-HCl (pH8.8)、Acr-Bis (29:1)、SDS等1]。本产品使用便捷。本下层胶预混液已经把SDS-PAGE凝胶下层胶成分预混合好，使用前仅需加入适量凝胶聚合催化剂(过硫酸铵或其替代物，如Ammonium persulfate substitute )和TEMED 即可简单快速地完成下层胶的配制。本产品适用于一步法配胶，快速高效。本产品优化了配方，灌注下层胶(即分离胶)后，无需液封可直接加入上层胶(又称浓缩胶、堆积胶或积层胶)，一步完成制胶，节约配胶时间。配合阿拉丁SDS-PAGE一步法红色或蓝色上层胶预混液使用效果更佳，轻松超快速完成相应浓度SDS-PAGE凝胶的配制。也可以直接选购相应浓度SDS-PAGE凝胶一步法超快速配制试剂盒(红色上层胶) ，使用更方便。本产品配制的SDS-PAGE凝胶，具有优良的分离效果。电泳后蛋白条带平整、清晰、细腻、锐利，特别是小分子蛋白质条带更清晰。阿拉丁生产的SDS-PAGE一步法下层胶预混液系列产品有6%、8%、10%、12%和15%共5种常见浓度供您选择，如需要配制其它浓度凝胶，可以考虑购买SDS-PAGE凝胶一步法快速配制试剂盒(红色上层胶) 、SDS-PAGE凝胶一步法配制试剂盒(红色上层胶) ，或单独购买Acr-Bis (29:1) 、10% SDS 和Tris-HCl (pH8.8) 等进行各种不同浓度SDS-PAGE凝胶的配制。 本产品的250ml包装约可配制30-60块常规大小的相应浓度SDS-PAGE凝胶的下层胶，500ml包装约可配制60-120块常规大小的相应浓度SDS-PAGE凝胶的下层胶。具体可以配制的凝胶数量和凝胶的厚薄以及凝胶的大小有关。4ºC避光保存，一年有效。进行下层胶配制时，需要另外准备过硫酸铵或其替代物以及TEMED 。本产品含有Acr-Bis，对人体有毒，操作时请特别小心，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.根据目的蛋白的分子量大小选择合适浓度的SDS-PAGE的下层胶(即分离胶)。不同浓度的SDS-PAGE分离胶的最佳分离范围如下： SDS-PAGE分离胶浓度 最佳分离范围 6%胶 50-150kDa 8%胶 30-90kDa 10%胶 20-80kDa 12%胶 12-60kDa 15%胶 10-40kDa 2.称取适量过硫酸铵或其替代物，如ST005，用超纯水或其它高纯度的水配制10%溶液。过硫酸铵或其替代物配成溶液后容易失效，需注意存放及时间限制。超纯水推荐使用 Ultrapure Water 。3.配制一块常用PAGE胶(胶板宽度为10厘米)所需下层胶和上层胶体积(下层胶按6厘米高度计算，上层胶按1.5厘米高度计算，均含约0.3ml的冗余量)参见下表。 凝胶厚度 下层胶体积 上层胶体积 0.75mm 4.0ml 1.0ml 1.0mm 5.4ml 1.5ml 1.5mm 8.0ml 2.0ml 注：下层胶体积已包含适量冗余，请勿全部用于灌制下层胶，以免灌胶时上层胶高度不够。4.参考下表，在相应浓度的SDS-PAGE一步法下层胶预混液中，按照1%的比例加入相应量的10%过硫酸铵或其替代物溶液，按照0.04%的比例加入相应量的TEMED。例如10ml 10% SDS-PAGE一步法下层胶预混液中，加入100μl 10%过硫酸铵或其替代物溶液和4μl TEMED，适当混匀后倒入到制胶模具中。本下层胶灌注后，无需等待凝固，可直接将配制好的上层胶混合液紧贴玻璃板均匀轻柔注入，随后小心插入梳子等待凝固。上层胶可以自行配制或者选用阿拉丁的SDS-PAGE一步法红色或蓝色上层胶预混液进行配制。也可用蒸馏水或异丙醇封住液面，直至下层胶凝固充分后再进行SDS-PAGE上层胶的配制。通常10-30分钟内胶会凝固。注1：一步法直接加入上层胶时建议配制4%的上层胶以达到更好的分层效果，也可根据需求自行配制不同浓度的上层胶。注2：一步法直接加入上层胶混合液时一定要轻缓并左右平移加入，而不是集中在一个位置灌制，以免上层胶溶液冲入下层胶而破坏上下层胶分界线的平整度，建议使用1ml移液器缓慢、均匀、左右平移加入。注3：一步法直接加入上层胶混合液，上下层胶分界线的平整度可能会稍差于液封法，但不影响后续电泳结果。注4：具体的凝固时间和温度及光照有关，说明书中10%过硫酸铵或其替代物和TEMED的推荐用量是室温为25ºC时的用量。为达到与25ºC时相近的凝固时间，当室温低于25ºC时，可以适当同时加大10%过硫酸铵或其替代物和TEMED的用量，例如20ºC时建议用量是正常推荐用量的1.5倍，15ºC时建议用量是正常推荐用量的2倍。 成分 配制不同体积SDS-PAGE下层胶所需各成分的体积(毫升) SDS-PAGE一步法下层胶预混液 5 10 15 20 30 50 10%过硫酸铵或其替代物 0.05 0.1 0.15 0.2 0.3 0.5 TEMED 0.002 0.004 0.006 0.008 0.012 0.02 5.配制好的凝胶如果当天不能使用，可以在4ºC保存1-2天后使用，但通常新鲜配制凝胶的电泳效果更佳。6.具体的电泳及Western实验操作可以参考如下网页：https://www.aladdin-e.com。参考文献：1.Laemmli UK. Nature. 1970. 227(5259):680-5. Aladdin's SDS-PAGE One-step Resolving Gel Master Mix contains almost all reagents required for the simple and ultra-fast preparation of DS-PAGE resolving gels at concentrations of 6%, 8%, 10%, 12%, and 15%, except for polymerization catalysts such as ammonium persulfate (APS) and TEMED. It contains Tris-HCl (pH 8.8), Acr-Bis (29 Precautions ： Ammonium Persulfate or its substitute (, ST005) and TEMED are required but not provided in this product.This product contains Acr-Bis which is potentially neurotoxic. Please take effective measures to avoid direct contact or inhalation.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.According to the size of a target protein, decide an appropriate concentration of SDS-PAGE resolving gel. Please refer to the table below. SDS-PAGE Resolving Gel Concentration Optimal Separation Range 6% 50-150kDa 8% 30-90kDa 10% 20-80kDa 12% 12-60kDa 15% 10-40kDa 2.Prepare a 10% APS solution with ddH2O or other high-purity water (, ST872). Solution of APS or its substitute is prone to failure and should be prepared freshly or kept frozen for multiple uses. 3.Prepare the required volumes of resolving gel and stacking gel. For a gel plate with a width of 10cm, please refer to the table below to prepare a resolving gel with a height of 6cm and a stacking gel with a height of 1.5cm. Gel thickness Volume of the resolving gel Volume of the stacking gel 0.75mm 4.0ml 1.0ml 1.0mm 5.4ml 1.5ml 1.5mm 8.0ml 2.0ml Note: Do not cast too much resolving gel. Otherwise, there will be no sufficient space for the stacking gel.4.Prepare the resolving gel according to the table below. For example, add 100μl of 10% ammonium persulfate and 4μl of TEMED into 10ml of 10% SDS-PAGE Resolving Gel Master Mix, mix well, and immediately pour into the gel cassette. Afterwards, directly and gently add the stacking gel onto the the top of the resolving gel. Insert the comb and leave at room temperature (~25℃) until fully solidified. The stacking gel can be prepared using ’s SDS-PAGE One-step Red or Blue Stacking Gel Master Mix . Alternatively, before preparing the stacking gel, overlay the resolving gel with the top layer of water-saturated butanol, isopropanol, 0.1% SDS or distilled water to allow for gel polymerization for 10-30 minutes.Note 1: It is recommended to prepare a 4% stacking gel to achieve better layering effect. Other concentrations of stacking gel also can be used as appropriate.Note 2: When adding the stacking gel, it is important to gently and slowly add it with a 1ml pipette while moving it left and right, instead of pouring it in one spot. This is to prevent the stacking gel from rushing into the resolving gel and disrupting the smoothness of the gel interface.Note 3: When directly adding the stacking gel for one-step method, the smoothness of the gel interface may be slightly worse compared to the liquid sealing method, but it does not affect the electrophoresis results.Note 4: The polymeriztion time varies depending on temperature and light conditions. The recommended amount of 10% APS and TEMED is for use at room temperature (25℃). At temperature lower than 25℃, it may be necessary to increase the amount of 10% APS and TEMED. For example, we recommend using 1.5 times the normal amount at 20℃, and 2 times the normal amount at 15℃. Reagents Volume of each component (ml) required for different volumes of SDS-PAGE resolving gel 10% SDS-PAGE One-step Resolving Gel Master Mix 5 10 15 20 30 50 10% APS or its substitute 0.05 0.1 0.15 0.2 0.3 0.5 TEMED 0.002 0.004 0.006 0.008 0.012 0.02 5.If the prepared gel is not to be used on the same day, it can be stored at 4℃ for 1-2 days. However, we recommend using freshly prepared gels to get better resolution effect.6.For electrophoresis and Western blot procedures, please refer to https://www.aladdin-e.com.References：1.Laemmli UK. Nature. 1970. 227(5259):680-5.