![SMOBIO/[PM5200] ExcelBand™ 3-color Pre-stained Protein Ladder, Broad Range (3.5-245 kDa), 250 μl x 2/Broad Range (3.5-245 kDa), 250 μl x 2/PM5200](images/SMOBIO/image.jpg)
Description
The PM5200 3-color Pre-Stained Protein Ladder Broad Range is a ready-to-use three-color protein standard with 15 pre-stained proteins covering a wide range of molecular weights from 5 to 245 kDa in Tris-Glycine Buffer (3.5 to 235 kDa in Bis-Tris (MOPS) buffer and 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 PM5200 3-color Pre-Stained Protein Ladder Broad 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, PM5200 ExcelBand™ 3-color Pre-Stained Protein Ladder Broad Range resolves 15 major bands in SDS-PAGE (Bis-Tris gel, MES buffer) and after Western blotting to nitrocellulose membrane.
Storage
4°C for 3 months-20°C for 24 months
Specification
Cat. No. | PM5200 |
Series Name | ExcelBand™ |
Product Size | 2 x 250 μl |
MW Range | 5 – 245 kDa |
Band Number | 15 |
Band Color | Red/Green/Blue |
Markered Bands | 75, 40, 20 kDa |
Manual
Manual_PM5200_ExcelBand™ 3-color Pre-stained Protein Ladder, Broad Range
SDS
SDS_PM5200
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 waterUnraveling the novel effects of aroma from small molecules in preventing hen egg white lysozyme amyloid fibril formation
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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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IgE是确诊过敏的唯一临床指标，人体的免疫细胞目前区分为三类，分别是TH1、TH2和Treg。在健康状态下，TH1和TH2会互相平衡，且共同受到Treg调控。当Treg调控能力不足时或接触到某些蛋白质或细小分子（尘螨花粉或海鲜等食物后，使TH2过度活化，导致 TH2细胞激素分泌量过高，就会帮助B细胞制造较多的过敏抗体IGE，因而出现过敏症状，康敏元抗过敏益生菌主要可调控因过敏而反应过高的TH2型细胞激素分泌量，进而调节免疫细胞活性平衡，可通过增进TH1型免疫反应来调控因过敏而反应过度的TH2型免疫反应的方法。
【过敏性咳嗽血清学IGE检查】
以过敏患者血清作为实验材料的本外试验方法称为血清学试验。其它体液如炎症部位分泌物、渗出物、灌洗液也可采用相同的实验方法进行检测。主要检测项目有总IgE和特异性IgE，即过敏原特异性IgE。
【什么是总IgE？】
IgE即免疫球蛋白E，是I型变态反应病如过敏性鼻炎、过敏性哮喘、异位性皮炎、湿疹、急慢性荨麻疹发病机制中起主要作用的免疫分子，因而在过敏反应的免疫学实验诊断中是首选的检测项目。总IgE是过敏性疾病的特异性检查项目，IgE水平增高提示I型变态反应病的可能性大，但不能用于判断过敏原。
【IgE的特点】
IgE是血清浓度最低的免疫球蛋白 ，只有血清中IgG浓度的万分之一。IgE对热不稳定，是半衰期最短一的免疫球蛋白 ，只有2.8天，与细胞表面结合的IgE半衰期稍长，8~14天，IgE由变应原入侵部位（鼻咽、支气管、胃肠道）的黏膜固有层中的浆细胞合成。在各类免疫球蛋白中，IgE是合成率最低、分解率最高的。属于亲细胞抗体，过敏体质者的胎儿脐带血中IgE浓度可能升高，检测脐血中IgE浓度可用于评估胎儿过敏体质的可能性。
【IgE检测方法】
通常用ELISA方法检测总IgE。由于血清IgE浓度很低，一般酶免疫试验方法的敏感性不足以检出血清IgE，现在常规实验室检测血清IgE的试剂盒采用生物素——抗生物素蛋白 放大的ELISA。试剂盒中所含用于制定标准曲线的IgE标准品和检测结果的IgE浓度单位与其它免疫球蛋白 不同，不是用mg/L表示，而是用u/ml或ku/l表示。
【IgE的正常值（参考范围）】：
血清IgE水平在正常人群中呈偏态分布，即多数人为0或接近于0，IgE水平越高的人数越少。因此计算平均值时应计算几何平均值才能反映其真实情况，即用对数转换后其分布才能近似正态分布。
健康人群血清IgE水平与年龄关系较大，小儿和老年人的IgE水平低于成年人。新生儿血清中IgE水平很低，接近于零。随年龄增长，IgE水平也不断升高，5~7岁后接近正常人水平。按Pharmacia公司提供的参考范围，1个月以内<12KU/L,1岁<11KU/L,2~4岁<33KU/L,5岁以上至成人<85KU/L.
过敏性疾病患者的血清IgE水平可达2000~8000KU/L,当IgE水平高于2000KU/L时应考虑寄生虫感染.
有时血清总IgE水平检测结果为0或参考范围内低值,并不能排除过敏性疾病的可能,须结合临床表现和血清特异性IgE检测结果进行判断.
【什么是特异性IgE检测(sIgE）？】
通常所称的过敏原检测，并非真正检测血液样本中的过敏原分子，而是间接地检测其中针对某种过敏原的特异性IgE分子，特异性IgE检测实际上是检测过敏原特异性IgE，即检测样本中针对某种变应原的特异性IgE，从而间接地判断患者是否对某种过敏原过敏。
环境中常见的过敏原包括以下类别：
寄生虫和微生物：各种螨类（屋尘螨和粉尘螨等）、各种真菌（点青霉、烟曲霉、分枝孢霉、交连孢霉等）、蟑螂。
植物花粉：各种草花粉（豚草、葎草、蒿草）、各种树花粉（桑树、柏树、悬铃木、桦树、榆树、柳树、杨树等）。
动物皮毛：猫、狗、马、鸽子等动物的毛和皮屑。
2006年，由蔡宗建（董事长）和池元（首席执行官），于福建福州（总部）成立“福州天盟数码有限公司”。
2009年，于新加坡成立“IGG Singapore Pte. Ltd.”（前身为“Skyunion Pte. Ltd.”）。
IGG主要专注于海外运营，截至2011年，IGG将20多款国产端游及页游推向海外市场，在全球拥有2000多万注册用户。鉴于在海外市场取得的良好成绩，IGG相继开发了《百年战争》、《泰坦战争》，准备凭借这两款产品进军国内市场。但国内的玩家对这两款网游并不买账，在国内表现不佳，几乎让蔡宗建赔光了老本，2011年底，IGG连员工的年终奖都没有发。由于这次惨痛的失败，蔡宗建不得不放弃国内市场。
2012年，IGG与facebook合作，推出了《Galaxy Online2》（星际文明2）。
2013年IGG投入了移动游戏市场，截至到2013年已经推出了9款手机游戏，截至2013年5月31日，IGG手机游戏日活跃用户的平均收益为0.08美元，平均日活跃用户为31.7万人。
2013年10月18日，蔡宗建带领下的IGG在港交所成功上市，公司市值达40.2亿港元。向左转|向右转
文章原文链接http://www.jbc.org/content/282/23/16776.long
本试验根据《中国药典》2015年版紫外分光光度法吸收系数法进行制定，本试验方法采用的方法学验证内容如下：
1.线性及范围
取IgG标准品配制5个不同浓度被测样品，并进行测定，绘制标准曲线，得到回归方程及范围。
2.准确度
已回收率对准确度进行验证。以已知含量的IgG标准品配制供试品，配制三个浓度，每个浓度平均测定三次。计算回收率。
3.精密度
IgG标准品配制供试品，平行测定六次。
4.溶液稳定性试验
至少持续2h的溶液稳定性考察。
5.干扰试验
空白溶液中加入辅料，在280nm处测定吸收，确定辅料是否对样品的吸收造成干扰。
以上，请大神指正。
补充问题，在专属性验证上，仅做辅料干扰是不是太少了？是否再进行强降解实验呢？因为其中包含了蛋白质A、外源性DNA及宿主细胞蛋白残留。我是否应该针对这三种杂质进行专属性试验？该如何进行呢？
风疹病毒抗体IgM阳性才需要治疗。
