Description
The PM2700 ExcelBand™ 3-color Broad Range Protein Marker is a ready-to-use three-color protein standard with 13 pre-stained proteins covering a wide range of molecular weights from 5 to 245 kDa in Tris-Glycine buffer (3.5 kDa to 235 kDa in Bis-Tris (MOPS) buffer and Bis-Tris (MES) buffer). Proteins are covalently coupled with a blue chromophore except for two reference bands (one green and one red band at 25 kDa and 75 kDa, respectively) when separated on SDS-PAGE (Tris-Glycine buffer). The PM2700 ExcelBand™ 3-color Broad Range Protein Marker 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.
Two reference bands — 75 kDa (red) and 25 kDa (green)
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, PM2700 ExcelBand™ 3-color Broad Range Protein Marker resolves 13 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. | PM2700 |
Series Name | ExcelBand™ |
Product Size | 2 x 250 μl |
MW Range | 5 – 245 kDa |
Band Number | 13 |
Band Color | Red/Green/Blue |
Markered Bands | 25, 75 kDa |
Manual
Manual_PM2700_ExcelBand™ 3-color Broad Range Protein Marker
SDS
SDS_PM2700
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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指在光、热、高能辐射、机械力、超声波和交联剂等作用下,大分子链间通过化学键联结起来,形成网状或体形结构高分子的过程。橡胶的硫化、不饱和树脂的交联、环氧树脂的熟化等都是化学交联的例子。通过化学交联可改善聚合物的性能。如聚乙烯的化学交联可提高其强度和耐热性,又如皮革的鞣制过程是利用其蛋白质分子与甲醛作用,生成交联桥,以至失去溶解性。
聚丙烯酰胺凝胶有以下优点:
①聚丙烯酰胺凝胶是由丙烯酰胺和N,N'甲叉双丙烯酰胺聚合而成的大分子。凝胶有
格子是带有酰胺侧链的碳-碳聚合物,没有或很少带有离子的侧基,因而电渗作用比较小,不易和样品相互作用。
②由于聚丙烯酰胺凝胶是一种人工合成的物质,在聚合前可调节单体的浓度比,形成
不同程度交链结构,其空隙度可在一个较广的范围内变化,可以根据要分离物质分子
的大小,选择合适的凝胶成分,使之既有适宜的空隙度,又有比较好的机械性质。一
般说来,含丙烯酰胺7-7.5%的凝胶,机械性能适用于分离分子量范围不1万至100万物
质,1万以下的蛋白质则采用含丙烯酰胺15-30%的凝胶,而分子量特别大的可采用含
丙烯酰胺4%的凝胶,大孔胶易碎,小孔胶则难从管中取出,因此当丙烯酰胺的浓度增 加时可以减少双含丙烯酰胺,以改进凝胶的机械性能。
③在一定浓度范围聚丙烯酰胺对热稳定。凝胶无色透明,易观察,可用检测仪直接测定。
④丙烯酰胺是比较纯的化合物,可以精制,减少污染。合成聚丙烯酰胺凝胶的原料是丙烯酰胺和甲撑双丙烯酰胺。丙烯酰胺称单体,甲撑双
丙烯酰胺称交联剂,在水溶液中,单体和交联剂通过自由基引发的聚合反应形成凝 胶。
在聚丙烯酰胺凝胶形成的反应过程中,需要有催化剂参加,催化剂包括引发剂和
另速剂两部分。引发剂在凝胶形成中提供始自由基,通过自由基的传递,使丙烯酰胺
成为自由基,发动聚合反应,加速剂则可加快引发剂放自由基的速度。常用的引发剂 和加速剂的配伍如下表:
聚合反应催化剂配伍
引 发 剂 加 速 剂
(NH4)2S2O8 TEMED
(NH4)2S2O8DMAPN
核 黄 素 TEMED
注:(NH4)2S2O8,过硫酸胺 TEMED:N,N,N,N';四甲基乙二胺 DMAPN:β-二甲基胺基丙晴
用过硫酸铵引发的反应称化学聚合反应;用核黄素引发,需要强光照射反应液, 称光聚合反应。 聚丙烯酰胺聚合反应可受下列因素影响:
1、大气中氧能淬灭自由基,使聚合反应终止,所以在聚合过程中要使反应液与空气 隔绝。 2、某些材料如有机玻璃,能抑制聚合反应。
3、某些化学药物可以减慢反应速度,如赤血盐。 4、温度高聚合快,温展开
要用几种不同材料来做这个实验
还要自己设计试验方法步骤所用仪器用品!
实在很是头疼谢谢!
在许多的细胞生命活动中,例如DNA复制、mRNA转录与修饰以及病毒的感染等都涉及到DNA与蛋白质之间的相互作用的问题.
重组DNA技术的发展,人们已分离到了许多重要的基因.现在的关键问题是需要揭示环境因子及发育信号究竟是如何控制基因的转录活性.为此需要:
a、鉴定分析参与基因表达调控的DNA元件;
b、分离并鉴定这些顺式元件特异性结合的蛋白质因子;
这些问题的研究都涉及到DNA与蛋白质之间的相互作用.
研究DNA-蛋白质相互作用的实验方法主要包括:
a、凝胶阻滞实验; b、DNase 1 足迹实验;
c、甲基化干扰实验; d、体内足迹实验; f、拉下实验.
1。什么叫切断条件。是不是不能切断的话就不能释放出药物
2。透膜性是什么意思。是不是指如果药物要进入细胞内的话就要选有透膜性的交联剂
3。间臂长度是不是臂越长越好
4。iodinatable是什么意思
谢谢大家!
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多谢您的支持!
转谷氨酰胺酶(TG)、过氧化酶(POD)、多酚氧化酶(PPO)交联
戊二醛,便宜,但有毒性
EDC,无毒性,但较贵
SMCC ,DSS,DST MBS,SPDP
P酸+5碳糖+含氮碱基形成脱氧核酸【DNA】,通过转录+翻译【涉及到的有转移RNA,信使RNA】然后识别信使RNA上的碱基排列顺序,把密码子连接起来,同时转移RNA上核糖体脱落形成氨基酸,氨基酸的排列顺序