![SMOBIO/[PM5100] ExcelBand™ 3-color Pre-stained Protein Ladder, High Range (9-245 kDa), 250 μl x 2/High Range (9-245 kDa), 250 μl x 2/PM5100](images/SMOBIO/image.jpg)
Description
The PM5100 3-color Pre-Stained Protein Ladder High Range is a ready-to-use three-color protein standard with 14 pre-stained proteins covering a wide range of molecular weights from 10 to 245 kDa in Tris-Glycine Buffer (9 to 235 kDa in Bis-Tris (MOPS) buffer and 10 to 235 kDa in Bis-Tris (MES) buffer). Proteins are covalently coupled with different chromophores for easy identification of bands, with three reference proteins carrying enhanced intensity corresponding to a blue band at 20 kDa, green at 40 kDa, and red at 75 kDa, respectively, as separated on SDS-PAGE (Tris-Glycine buffer). The PM5100 3-color Pre-Stained Protein Ladder High Range is designed for monitoring protein separation during SDS-polyacrylamide gel electrophoresis, verification of Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) and for approximating the size of proteins.
Features
Ready-to-use — Premixed with a loading buffer for direct loading, no need to boil.
Three reference bands — 75 kDa (red), 40 kDa (green), and 20 kDa (blue)
Contents
Approximately 0.1~0.4 mg/ml of each protein in the buffer (20 mM Tris-phosphate (pH 7.5), 2% SDS, 0.2 mM DTT, 3.6 M urea, and 15% (v/v) glycerol).
Quality Control
Under suggested conditions, PM5100 ExcelBand™ 3-color Pre-Stained Protein Ladder High Range resolves 14 major bands in 15% SDS-PAGE (Tris-Glycine buffer) and after Western blotting to nitrocellulose membrane.
Storage
4°C for 3 months-20°C for 24 months
Specification
Cat. No. | PM5100 |
Series Name | ExcelBand™ |
Product Size | 2 x 250 μl |
MW Range | 10 – 245 kDa |
Band Number | 14 |
Band Color | Red/Green/Blue |
Markered Bands | 75, 40, 20 kDa |
Manual
Manual_PM5100_ExcelBand™ 3-color Pre-stained Protein Ladder, High Range
SDS
SDS_PM5100
Migration patterns and approximate MWs (kDa)
Why are there contrasting results in molecular weights after using different brands of protein markers?
A.Different proteins even with similar molecular weights would exhibit apparent disparity from the resulting SDS PAGE due to the difference in the composition of the protein’s amino acids (e.g. gelatin). The reason for the disparity is due to the amino acids composition that affects the binding of the protein and SDS. Therefore, we can say that protein marker is a handy tool to estimate molecular weight, but there is no absolute molecular weight standard.
B.While running SDS-PAGE, protein mobility can be affected by the composition of the buffer used, gel percentage, the voltage used, running time, as well as if there is a pre-run.
C.Another recommendation for high molecular weight proteins is to prolong the running time to clarify the relative location of bands.
Protein marker Retention Period: Mentioned -20°C and over 2 years. Is it available for 30 months or 36 months? Have you tested this period?
Yes, we have tested our PM2700. The results showed that the PM2700 is stable at -20℃ for at least two years. It has also shown strong performance for more than 36 months under our careful storage. However, we must only suggest a 2 year retention period for the following reasons: There may be a variation in the environment in storage, and improper use may lead to accumulated damage to the proteins and therefore reduce its retention period.
How many times of freezing and thawing are available for protein markers? If it uses 5 μL per load, would the total usage quantity be 50 times x 2 (250 μL x 2 tube)?
Yes, 100 uses (5 μL each time) can be expected if freezing and thawing are conducted carefully and properly at the appropriate temperature. Before each use, make sure the protein marker is thoroughly thawed.
Do you have data comparison for protein molecular weight’s precision with other protein markers?
Yes. Usually, pre-stained marker is written on “estimated molecular weight” for caution. It is known that the analysis of protein size by an SDS-PAGE is only for “estimation” because of the intrinsic variation of amino acid composition in all proteins including stained and non-stained ones. For example, a protein which is highly hydrophilic might show a particular higher position in the SDS-PAGE analysis when compared to a hydrophobic one. We did compare the migration patterns of SMOBIO’s Protein Markers with other brands, and we concluded that it was difficult to define “precision” due to the reasons mentioned above. Therefore, in the product description, we suggest our users to calibrate the MW against their interested proteins. Although it is impossible to define "precision" for molecular weight of proteins in SDS-PAGE, we did compare the migration pattern of pre-stained markers with unstained protein marker (Invitrogen MARK12) for calibration. It is concluded that the estimated molecular weight of SMOBIO’s pre-stained marker shows a curve matching well with that of unstained native proteins (MARK12), representing a good estimation of the MW of each pre-stained protein in the SDS-PAGE analysis.
Will SMOBIO’s Protein Markers/Ladder be washed out during Western blotting process?
SMOBIO’s Protein Markers/Ladder will be only slightly washed out during Western blotting process. However, the excess of Tween-20 (more than 0.2%) in washing buffer will affect SMOBIO’s Protein Markers/Ladder on the transfer membrane.
Here are suggestions for Western blotting process:1. Transfer SMOBIO’s Protein Markers/Ladder to membrane with transfer buffer containing 20% methanol to fix SMOBIO’s Protein Markers/Ladder on membrane. 2. Wash membrane with PBS or TBS containing less than 0.1% Tween-20.
Will SMOBIO’s Protein Markers/Ladder be affected by the stripping/deprobing process with the presence of β-Mercaptoethanol (β-ME)?
In normal circumstances, the presence of βME during the stripping/deprobing process will only slightly affect SMOBIO’s Protein Markers/Ladder. However, the presence of Tween-20 on PVDF membrane during the stripping/deprobing process has adverse effects on SMOBIO’s Protein Markers/Ladder.
Here are suggestions for Western stripping/deprobing process:
1. Wash the PVDF membrane in methanol for 5~10 minutes prior to the stripping/deprobing process to mitigate the adverse effect of Tween-20.2. Recommended stripping buffer (for 1 L): 15 g glycine, 1 g SDS, 10 mL Tween 20. Dissolve in 800 mL distilled water. Adjust pH to 2.2 Bring volume up to 1 L with distilled waterEfficient RNA drug delivery using red blood cell extracellular vesicles
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ExcelBand™ Protein Markers
Ready-to-use— premixed with a loading buffer for direct loading, no need to boil
Broad range— 310 kDa to 5 kDa
Pre-stained bands — for monitoring protein separation during electrophoresis and Western blotting transferring efficiency on membrane
Enhanced bands— for quick reference
YesBlot™ Western Marker I
Ready-to-use — no need of mixing or heating before sample loading
Direct visualization — 10 IgG-binding proteins for direct visualization on Western blots
Pre-stained bands — 4 pre-stained proteins for monitoring protein separation during electrophoresis and Western blotting transferring efficiency on membrane
Wide range — 10 clear bands from 15 to 200 kDa for size estimation
Quick reference — two enhanced bands (30 and 80 kDa)
Q-PAGE™ Precast Gels
User-friendly gel cassette:
Numbered and framed wells for sample loading
Labeled warning sign and green tape as reminder
Enhanced gel performance:
Enhanced gel electrophoresis speed
Better band separation
Stable for shipping at ambient temperature
Easy compatibility:
Available as homogeneous and adjusted gradient gels for a wide range of protein separation.
Compatible with most popular protein electrophoresis systems
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液体的一抗加50%甘油，-20度也不会冻结，可保存3-5年；
经常使用时取出一小份，4度可使用半年之久；
粉末状的二抗直接-20或-80度贮藏就行了。
我的实验是这样的，先纯化质粒，然后做酶切，再纯化DNA。
最后一步其实也可以用切胶回收试剂盒，我怕做不好，所以想选用DNA纯化试剂盒，但不知跟质粒DNA提取试剂盒是否有区别，因为说明书上说可以用Invitrogen的K210001.我搜索看了一席啊，觉得那个是提取质粒的小提啊。所以产生这个疑问，请大侠告知DNA纯化试剂盒与质粒DNA提取试剂盒这两者之间有区别吗？
用哪个公司的DNA纯化试剂盒比较好。谢谢啦！
2.磁珠法提取DNA试剂盒
3.DNA提取试剂盒（离心柱法）
4.磁珠法RNA提取试剂盒
5.RNA提取试剂盒（离心柱法）
6.磁珠法植物基因组DNA提取试剂盒
7.反转录试剂盒
8.胶回收试剂盒
9.PCR纯化试剂盒
10.病毒核酸提取试剂盒（磁珠法）
11.土壤基因组DNA提取试剂盒
12.法医样本DNA提取试剂盒（磁珠法）等 1.elisa
常见elisa试剂盒有：
特种蛋白检测elisa试剂盒
如免疫球蛋白,抗链O-aso,类风湿因子RF,C反应蛋白,微量白蛋白,β-2微球蛋白human,铁蛋白,转铁蛋白transferrin等等
肿瘤标志物检测elisa试剂盒：
如肿瘤标志物，组织多肽抗原，肿瘤相关因子，胰腺癌，直肠癌，细胞角蛋白片段，胃肠癌，铁蛋白，糖链抗原，神经特异性稀醇化酶，上皮膜抗原，乳腺癌，人抗小鼠抗体，前列腺，甲胎蛋白，肝癌，甲基苯丙胺，大肠癌，肺癌，大小便隐血检测，癌胚抗原，β-2微球蛋白等等。
食品安全检验elisa试剂盒：
食品中的激素、药物、霉菌毒素、过敏原残留、转基因产品的检测试剂盒，以及微生物、维生素等的检测产品。
植物病毒、细菌、真菌、植物激素和转基因作物的农业诊断试剂盒
动植物疾病诊断类如猪、牛、羊、马等家畜和禽类以及宠物类检测试剂盒
传染病检测elisa试剂盒
如幽门螺杆菌,乙脑,乙肝,丙肝,丁肝,戊肝,庚肝,衣原体,性病,腺病毒,微小病毒B19,天疱疮,水痘-带状疱疹病毒,生殖支原体,伤寒,沙眼,腮腺炎,人型支原体,麻疹,轮状病毒,流行性出血热,淋球菌,莱姆病,柯萨奇,抗解尿支原体,军团菌,结核,胶原,尖锐湿疣,甲肝,脊髓灰质炎,急性胰腺炎尿胰蛋白酶,霍乱,呼吸道合胞病毒,肝吸虫,副流感,肺炎,带状疱疹,传染性单核细胞增多症,层粘蛋白,布鲁氏杆菌,百日咳,白喉,艾柯病毒,EB病毒,A族链球菌等等。
2.免疫共沉淀试剂盒
3.化学发光试剂盒
4.免疫组化试剂盒
5.放射免疫试剂盒
6.免疫荧光试剂盒 1.细胞凋亡试剂盒
2.葡萄糖检测试剂盒
3.支原体检测试剂盒
四、试剂盒使用示例
试剂盒的产生正是为了使实验人员能够摆脱繁重的试剂配制及优化过程，所以试剂盒中一般配备有相应的使用说明书，用户按照说明书不需或只需少量的优化即可得到满意的结果。
⑴试剂盒使用说明书
说明书一般包括公司标志及名称、试剂盒名称、试剂盒组成、保质期、使用领域、使用方法等项目
说明书格式如右图所示：
①一般在页眉页脚处为公司的名称及标志；
②接下来为试剂盒的名称；
③试剂盒组成中应为试剂盒中的所有内容，为了简洁明了，一般以表格的形式表现；
④操作步骤是试剂盒说明书中最重要的部分，内容应准确、简洁、易懂，不能包含带有歧义的句子，更不能含糊其辞，排版上操作步骤应将复杂的步骤拆解，使人一目了然。可以说操作步骤为试剂盒的灵魂。 试剂盒内容（以离心柱型为例）：
一般包括：裂解液RL
去蛋白液RW1
漂洗液RW
RNase free ddH2O
吸附柱（RNase free）
过滤柱（RNase free）
缓冲液
离心管（RNase free）
收集管（RNase free）
此外，还配有一份试剂盒使用说明书，根据不同的生产商，可能还配有不同的试剂，如：DNA酶、溶菌酶等。
使用时一定要根据自己的实验需要购买专用提取试剂盒， 如果不是专用试剂盒，或许能提出RNA，但不能确保其质量以及完整性，会影响RT-PCR、Northern blot、Dot blot、Real time RT PCR、芯片分析、polyA 筛选、体外翻译、RNase 保护分析、分子克隆等后续实验的结果。
当前提取效果好的是QIANGEN公司生产的RNA提取试剂盒，但其售价较高，单次实验费用花费太大，对精度要求不高的实验没有必要购买，当然还有其它的进口试剂盒，但是均存在价格高、订货周期长的问题（如果碰上国内无货的时候，要从国外发货，会等很长一段时间）。普通的提取实验用国产的试剂盒就足以，价格不高，基本上各地均有现货，如天根（目前国内生产的试剂盒中销售面比较广的一种）等，其价格比进口试剂盒要便宜许多，且效果还不错，性价比还可以。
当然，就RNA的提取而言，不一定试剂盒的提取效果就非常好，其实采用一些经典的RNA提取方法，效果也很不错，如：TRIZOL、EDTA等，只是试剂盒使用方便，经典的方法操作复杂一些。向左转|向右转
C、如果检测的指标数目超过了流式细胞仪的通道，怎么办？如果需要做5个指标，而流式细胞仪只能做4个通道，首先将指标归成2类，再将样本分成2份，分别检测。比如需要同时检测CD3、CD4、CD8、IL-4和IFN-gamma，那么将样本分成两份，第1份加CD3、CD4、CD8和IL-4，而第2份加CD3、CD4、CD8和IFN-gamma。
二、同型对照（IsotypyControl）1、为什么要用同型对照？同型对照是使用与一抗相同种属来源、相同亚型、相同剂量和相同的免疫球蛋白及亚型的免疫球蛋白，用于消除由于抗体非特异性与细胞结合而产生的背景染色。同型对照是真正意思上的阴性对照，它不但可以用来设定流式细胞仪的电压，而且还可以帮忙省去昂贵与繁琐的重组细胞因子竞争封闭步骤。在流式细胞仪上样前，染色方式如下：样本管：一抗＋样本同型对照管：同型对照＋样本
2、如何选择同型对照？一般选择与一抗的成分完全相同种属来源、相同亚型和亚链、相同荧光标记的抗体，比如抗人的CD56APC标记的抗体，货号是555518,成份是MouseIgG1,κ。那么它的同型对照应该是APC标记的MouseIgG1,κ，货号是555751。注意同型对照的成份跟它的名称是相同的.如果是抗体的组合形式是纯化的一抗＋荧光标记的二抗，那么应该选择一抗的同型对照。比如CDw125的纯化抗体，货号是555901，成分是MouseIgG1,κ。它的同型对照是纯化的MouseIgG1,κ，货号是555746。它的二抗PE标记的抗小鼠IgG1,货号是550083。那么染色方式如下：样本管：纯化的CDw25＋PE标记的抗MouseIgG1＋样本同型对照管：纯化的同型对照＋PE标记的抗MouseIgG1＋样本
如果没有找到适合的同型对照，在保持来源相同、荧光标记相同、免疫球蛋白类别相同条件下，那么优先选择的顺序应该是亚链不同－－亚型不同－－相同类别。比如，某种的抗体的来源是MouseIgG2а,κ。如果在同型对照表里面找到不到完全相同的同型对照，那么优先选择相同荧光标记的MouseIgG2а为同型对照。如果也没有找到MouseIgG2а，那么优先选择相同荧光标记的MouseIgG2b作为同型对照。如果最后还是没有找到MouseIgG2b，那么选者相同荧光标记的MouseIgG2作为同型对照。
3、如何查找同型对照？同型对照在BD英文目录中或者BD网站上跟它对照的抗体的位置是一致的。BD目录中，抗人的抗体的同型对照都在HUMAN章节后面。抗小鼠的抗体的同型对照在MOUSE章节后面，非人类灵长类的抗体同型对照在NONHUMANPRIMATE章节后面。由于抗大鼠的抗体非常少，大部分抗体都是小鼠来源的，所以它的同型对照跟小鼠的一样，放在MOUSE章节后面。胞内细胞因子、趋化因子、补体、炎症介质及其受体的同型对照放在其章节的后面（注意：人的胞内细胞因子的同型对照跟人的表面标志的同型对照是不在一起的）。Phosflow的同型对照在Phosflow的介绍后面。比如，比如抗人的CD56APC标记的抗体，货号是555518，在06版BD目录的169页，成份是MouseIgG1,κ。那么它的同型对照应该是在HUMAN章节中MouseImmunoglobulinIsotype表格中，第191页，APC标记的MouseIgG1,κ，货号是555751。
4、哪些客户需要使用同型对照？A对流式不是非常熟悉的客户。B做胞内流式客户，比如胞内细胞因子、趋化因子和信号蛋白等。C使用不常见的标志或者特异性不高的标志的客户，因为客户不熟悉不常见标志的表达情况，根据同型对照可以判断阳性细胞的位置。一些特异性不高的抗体，如多抗，非特异性结合非常多，使用同型对照可排除假阳性。D对流式非常熟悉又使用常用的表面标志的客户一般可以不使用同型对照。
5、为什么有时候同型对照的表达会比抗体的表达高？首先需要检查同型对照的抗体是否加错类型、剂量等。其次，因为有些标志在细胞的表面或者胞内表达不高，而跟其类似的抗原非常多，那么加入同型对照时，同型对照非特异性的结合会超过抗体的特异性，所以会造成这种现象，在细胞表面时如图所示。这个时候推荐使用其他阴性对照，如空白对照等。
三、补偿微球流式细胞仪的荧光补偿隔一段时间之后需要调整到适合的位置。如果荧光补偿没有调节好，会导致细胞分群不明显或者流式检测得到的结果图非常不漂亮，而且会影响到检测结果的不真实。荧光补偿的调节对初学者来说非常不容易掌握，同时，补偿调节是流式细胞术中比较难掌握的操作。尤其是一些不常见的标志检测时，需要经验非常丰富的流式操作者根据多年的经验判断调节补偿，才可以得到比较好的数据和图片。那么，现在不需要这么麻烦。因为补偿微球让一切变得更简单。BDCompBeads是专用于流式细胞仪多色分析的荧光补偿调整微球。它的实用性是在于克服了以往普通的做三色以上的流式分析时补偿难调、耗费样本（往往用待测样本单标来进行补偿调节）的缺点。它本身不携带任何荧光，借助于与客户自己的特异性荧光抗体孵育结合来调节补偿。它不但能象待测的样本细胞一样在同样的实验条件下固定、破膜等，操作起来很简单，灵敏度高，一致性好，使补偿更轻松更准确，而且最难得的是它最适合于多色分析（如五色、六色、七色等）和复合荧光素（如PE-Cy7、APC-Cy7等），因为这时的补偿往往难度较大。各型号的流式仪器均能使用。Compbeads使用后可以将流式的条件保存，方便下次做同样的荧光染色搭配时参考用。
双抗无论是在免疫学还是细胞操作，细胞培养中都不与二抗等同。双抗可以是抗一种抗原的两个抗体，去双抗夹心法。也可以是抗不同类型反应的作用物，如抗凝剂素。一般指两种抗生素。
