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多角度激光光散射仪与凝胶渗透色谱联用 技术及文摘

发布时间:2013-01-02   点击次数:4465次

 

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多角度激光光散射仪与凝胶渗透色谱联用

技术及文摘

 

MALLS/GPC(SEC)

 

 

 

 

 

 

 

WYATT TECHNOLOGY CORPORTION

(BEIJING OFFICE)

地址:北京西直门北大街58号 金晖嘉园7-2302

      美国怀雅特技术公司北京代表处

:100082

:8610-82292806

8610-82290337

 

多角度激光光散射仪与凝胶渗透色谱联用技术

Wyatt Technology Corp. 

一 前言

近十几年来,光散射技术(Light scattering)在高分子特征分析领域的应用得到了迅速的发展。将光散射技术和凝胶渗透色谱(Gel Permeation Chromatography, GPC)或尺寸排阻色谱(Size Exclusion Chromatography, SEC)分离技术相结合,不但可以测得大分子的分子量,分子旋转半径与第二维里系数,还可测得分子量分布,分辨分子量大小不同的族群,判断溶液中大分子的构象,分枝率及聚集态等。

作为多角度激光光散射仪的---Wyatt Technology Corp. 结合多个技术生产的DAWN系列产品在国内外有着广泛而的用户基础;的性能、可靠的质量、稳定的服务,Wyatt的产品一直是广大科技工作者的产品。

 

二 光散射简介

   早在十九世纪初,人们就开始对光散射原理进行研究。自六十年代激光被发明以来,光散射的原理与技术便得以迅速发展,至今已成为检测微小粒子形状,粒径大小,分子量,界面电位及粒子间效应的重要工具。随着电脑技术的日新月异,许多过去需花费数小时甚至数日才能完成的实验,如今只需数分钟即可完成,而其准确性及重现性也大幅度提高了。

   光散射现象,如图1 所示,当一束光通过一间充满烟雾的房间就会产生散射。利用在不同角度,不同时间所测得的光散射强度,再借助各种光学理论及软件,硬件设备,就可以测得微粒的许多特性。

 

入射光

 

 

 

 

散射光

图1光散射现象

 

在光散射发展的历程中,以下是一些具有代表性的人物:

▲James Clerk Maxwell (1833-1879)

解释了光是一种电磁波,并正确地计算出光的速度。

  • Lord Rayleigh(1842-1919)

研究了远小于波长的微粒散射现象,发现了散射强度与波长的四次方成反比,并解释了蓝天被太阳光穿透大气层所产生的散射现象。

  • Abert Einstein(1879-1955)

研究了液体的光散射现象。

▲Chandrasekhara V.Raman (1888-1970)

印度籍物理大师,提出了Raman 效应,其著作多次发表于印度文期刊,直至第二次世界大战结束后才逐渐被人所知。

  • Peter Debye(1884-1966)

延续了Einstein的理论,描述了分子溶解于溶剂中所产生的光散射现象,提出用Debye plot, 求得重量平均分子量Mw。

 

三 光散射理论

   激光照射到样品时,会在各个方向产生散射光,于是我们可以在一个角度或多个角度收集散射光的强度。

 

  1. 光散射所透露的信息

在任何方向的光散射强度与分子量和溶液的浓度成正比;散射光角度的变化与分子的尺寸大小有关。当分子小于10nm时,各个角度的散射强度都相同;当分子介于10至30nm时,散射强度则由低角度向高角度呈直线下降的趋势;而当分子大于30nm 时,散射强度则随角度增大呈曲线下降的趋势。

 

  1. 基本理论

由Maxwell,Einstein,Debye及 Zimm 等人陆续发展起来,有关溶剂中分子量的光散射现象可由下列公式表达:

 

 

 

 

(1)

式中:

常数K*=4π2(dn/dc2n02(NAλ04

n0是溶剂的折光指数。

         NA是阿佛加德罗常数。

λ0是入射光的波长。

dn/dc是溶液折射率与浓度变化的比值,它说明了随溶质浓度变化的溶液折光指数变化。

C是溶质分子的浓度(g/mol)。

Rθ)是单个角度的散射光(大于溶剂的散射光数量)除以入射光强度所得的分数即不同角度光散射强度。

Mw是重均分子量。

A2 是第二维里系数

Pθ) 是光散射强度的函数

Pθ)代入式(1)展开得:

 

 

在上式中,Rθ)是测得值,K*c、λ0θ为输入值,均为已知值;而MwA2rg为未知值。

 

  1. Zimm Plot

将K* C/ Rθ对sin2(θ/2)+kc作图,可得到的Zimm曲线,如图2所示,其中K为调整横坐标的设定值。

 

 

 

 

 

 

 

 

 

 

                                      图2 Zimm Plot

θ→ O时,(2)式简化为

斜率即是A2

 

 

C→ 0时,(2)式简化为

斜率是rg2

 

θ→ OC→ 0,(2)式简化为

 

 

 

在纵座标上交点的倒数即为Mw。实验的方法为配制一组不同浓度的溶液,依次在不同的角度测量其散射光强度,由计算机程序按照上列的公式绘出Zimm Plot,并求得Mw,<rg2>及A2值,这是极少数能直接测得分子量的方法之一。但由于结果仅为单一平均值,因此较适用于成分单一,分布较窄的分子,对于分布较宽或有不同族群分布的样品,则较难看出全貌。

 

四 光散射与GPC/SEC

GPC/SEC可以将溶剂中的分子按重量或尺寸大小依次洗脱出来。利用此项技术将光散射仪器与GPC/SEC联用,除了可以分出不同的族群,还可测得不同族群的分布,并且不需要另外标准样品做标准曲线。由于光散射信号直接与分子量大小有关,因此可以直接测出重均分子量,并获得其它许多有关的信息。

 

 

 

 

 

 

 

 

 

 

 

 

 

图4 光散射强度与GPC层析图

 

Chromatography with LS Set-up

 

 

 

 

 

 

 

 

 

 

 

 

 

 

图5 光散射强度与GPC/SEC联用

 

1 Debye plot

通常GPC/SEC 的样品注射浓度就很低,再经过色谱柱得到进一步的稀释,图4中光散射信号上的任何一点,其浓度都极低(趋近于零)。根据公式,当2 A2C → 0,

 

 

 

将K* C/ Rθ对sin2(θ/2)+kc作图6,其纵坐标交点即为1/Mw,由直线的斜率可得到<rg2>,图4光散射信号的每点都可以得到上述结果,由此可以求得分子量及旋转半径<rg2>的分布,如图8所示:

 

 

 

 

 

 

 

 

 

 

 

 

图6 Debye plot

 

 

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图7 积分分子量

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

图8 分子量对洗脱体积作图

 

 

2 分子形状  

  不论是分布较宽或是多峰分布的样品,皆可通过测量分子量及分子旋转半径得到分子形状的数据。

  球形分子

  ri3 ∞ Mi→log ri = k + 1/3 log Mi

无规则线团状分子

  ri2 ∞ Mi→log ri = k + 1/2 log Mi

棒状分子

ri1 ∞ Mi→log ri = k + 1/1 log Mi

 

 将log ri, log Mi作图,有直线的斜率可以获知分子的形状,如图9所示:

 

 

                              棒状(斜率=1)

 

                                               无规线团(斜率0.5-0.6)

                logrg

                                               球型(斜率=1/3)

                                                  

 

 

 

 logM

 

图9-1  M、rg与分子形状的关系

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图9-2 构型判断, rg对Mw作图。斜率为0.54 ± 0.01。表明分子是具有无规则线团构象的线性聚合物

 

 

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图9-3 构型判断,rg对Mw作图。斜率大于0.6,表明分子具有伸展结构。斜率为1.0,表明是棒状结构。

 

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图9-4 构型判断,rg对Mw作图。其U型曲线表明为典型的高支化度结构。

3 需要量多少个角度

   在低角度的时候,有杂质所产生的噪音信号干扰会很大,如图10所示,所以只取低角度,加上九十度两个角度,其误差就会相对很大;若只取九十度则只能求得分子量,无法测得旋转半径,所以zui起码要加上一组高角度来修正,则误差会减少很多。

 

 

 

 

 

 

 

 

 

 

图10 杂质较多的GPC层析图

 

4 光散射仪器

zui基本的仪器mini DAWN TREOS如图11所示,在与入射光成45度、90度、135度角配置三组光电二极管检测器,同时检测不同角度的光散射强度,而激光经由样品槽的毛细管通道,样品槽为石英材质。

如果要增加测量角度,可以如DAWN HELEOS

在样品槽的两侧以不对称得方式增加检测器的数目,

如图12所示可高达18个角度之多。

 

 

 

图11 三个检测角度

五 应用

   光散射强度与分子的大小及分子量有直接的关系,而SEC/GPC能分离不同尺寸及分子量的分子,结合此两种特性,可以得到许多有用的信息,并广泛地应用于高分子,生化及动力学等研究领域。

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12 十八角度检测器

1 高分子聚合物和天然高分子的特性研究

利用多角度激光光散射系统(Multi-Angle Laser Light Scattering_MALLS) 结合SEC/GPC,不必依赖泵的流速,校正曲线及其它任何的假设,即可直接求得重均分子量及分子量分布等数据。

 

13 光散射强度,洗脱体积与角度作图

 

MALLS利用色谱柱分离出的样品在各个角度的光散射量(如图13),由RI 检测器得到的洗脱液浓度及dn/dc值,即可计算出各个切片的分子量。MALLS测得分子量所需的各种物性均可由实验直接求得,无需作任何假设。而GPC的色谱柱又有分离杂质的功能,可以避免传统的光散射需极小心准备样品的麻烦。图14显示高分子混合物经SEC分离后MALLS及RI的洗脱体积对照图。由此图看出RI 对大分子量浓度低的物质较不敏感,而对低分子量高浓度者较敏感。

 

 

 

 

 

 

 

 

 

 

图14 这是由ASTRA软件得到的miniDAWN(上)和Optilab示差检测器(下)信号。

1   BSA     67,000   64,300±700     1%    2   溶解酵素 14,300   14,600±300     1%

3   缓激肽   1,060    1,090±10      2%    4   亮氨酸脑啡肽 556  592±6          3%

 

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图15分子量对洗脱体积图,有四个明显的峰

 

2. 蛋白质及其聚合体

在各种工业应用中,决定蛋白质的特性不仅严格而且必要,例如在生化工程应用上,以蛋白质为基质的产品必须很纯而且无任何聚集存在。而测定蛋白质的分子量和是否有聚集态存在,光散射法是zui理想的工具之一。

以往,在水相中用低角度光散射测量法(LALLS)受到溶剂中不纯的物质干扰相当大。而MALLS的多角度测量大大降低了背景噪音的干扰,并能提供完整的信息和良好的重现性结果。图16显示蛋白质混合物的MALLS 和RI的信号。样品在0.1M NaCl 中含0.05M的磷酸盐缓冲液中进行RI为Wyatt Optilab 903,流速为0.1mL/min ,色谱柱为Shodex KW-803 和KW-804。虽然此样品为标准样品,但MALLS仍很清楚地检测到聚集现象,此现象在RI几乎无法辨认。

 

 

 

 

 

 

 

图16蛋白质洗脱体积及聚集体信号图    

 

3.分枝

高分子聚合物的分枝程度和分布是影响其物理和化学性质的一个重要因素。采用多角度激光光散射系统(MALLS)与GPC/SEC系统联用是*决定分枝系数gM的方法。虽然也有其他确定分枝的方法,但都不能直接且需要众多假设及“虚拟因子”。

由传统的RI或Viscometer (粘度检测器) 测定的高枝化分子的分子量与值有很大的差别,若欲做有效的色谱柱校正,则需以一系列与待测物成分相同的标准品作校正。若标准样品与待测物的成分或组分不同,则会产生很大的误差。例如分子量相同的球形高分子的洗脱时间比无规则线团状分子要长。

因为MALLS所求得分子量和大小为值,因此计算分枝系数gM不需要任何假设。由MALLS直接所求得的分子大小会直接影响分枝率。对分子量相同的长链状分子而言,其值越小,则分枝程度越大。分枝比的定义为分枝分子的旋转半径与长链分子的旋转半径之比,即gM=<r2>b/<r2>l由MALLS测得。

图17为由MALLS测得的分枝状和长链形的高分子(PS)的旋转半径和分子量对照图。由图中可看出,虽然其分子量相同,但分布明显不同。图18 为rg Mw做图。

 

 

 

 

 

 

 

 

 

 

 

 

 

 

图17 PS线形与枝化分子的对照图

 

 

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图18旋转半径对分子量图(分子构型图),可以看出样品(藻酸钠)在辐射后分子构型的变化。

 

6. 动力学/反应速率

MALLS还可以用于如抗原、抗体等反应迅速的溶液系统,粒子和蛋白质聚集现象的检测。因为MALLS 内部同时装有数个固定的检测器,所以不需移动任何仪器硬件来扫描样品,即可及时多方位同时捕捉反应速率的现象。使用MALLS,可研究抗原-抗体反应,反应发生时就可决定聚集粒子的大小。当改变温度,浓度或催化剂时,MALLS可记录下反应发生时,分子的特殊变化。

 

 

 

 

 

 

 

 

 

 

 

 

图19 浓度变化对蛋白聚集的影响

 

使用DAWN 检测器研究浓度对分子量为75KD单分子蛋白质聚集作用的影响。图19描述了这种特殊蛋白质从30ug/ml1mg/ml范围内得到的浓度相关性。如图所示,该蛋白质在低浓度作为单一分子而在浓度大于700ug/ml时聚集为六聚体。该结果与由gluteraldehyde高度交联技术所得结合完全相符。

 

 

 

 

 

 

 

 

 

 

 

 

 

图20 温度变化对PMMA分子量和大小的影响

 

7. 低分子量的测定

DAWN HELEOS或mini DAWN TREOS的固定光电二极管检测器可以捕捉到很微弱的光散射信号,使得低分子量的测定成为可能。使用DAWN 系列标准配制的任何一款激光器,都可以轻易地测量分子量低于2000D的聚合物,并具有相当的准确性。  

由于DAWN HELEOS 或mini DAWN TREOS具有三个以上的多角度同时捕捉散射信号的能力,即使极微弱的信号,如只比背景值略高的低分子量样品所散射出的信号也可以从不同的角度去捕捉,累计在一起就可以计算出相当准确的结果。这是单角度或者两角度检测器所无法做到的。

图21为分子量分别为580、1400及2000D 的聚苯乙烯样品分子量对洗脱体积的对应图。样品量浓度分别为7.1mg/ml,2.9mg/ml及2.2mg/ml。经ASTRA  软件分析得出如表2的平均分子量。

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

图21 低分子量样品与洗脱体积图

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

图22低分子量样品分子量图分布图

 

表2样品平均分子量

 

 

Sample Stated Mw

Low Scattering Mw

A

580

512±17

B

1400

1371±29

C

2000

2012±40

 

 

一般传统光散射仪给人们的印象是不易测得分子量较低的样品,甚至低于10000D就比较困难了。但是采用的多角度激光光散射仪就可以轻易且相当准确的测量几百D分子量的样品。

   

 

六 代表性文献

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  1. Design and Testing for a Nontagged F1-V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague   

B. S. Powell, G. P. Andrews, J. T. Enama, S. Jendrek, C. Bolt, P. Worsham, J. K. Pullen, W. Ribot, H. Hines, L. Smith, D. G. Heath, J. J. Adamovicz, "Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague", Biotechnology Progress 21(5), 1490-1510 (2005).

Summary: During discovery and testing of the F1-V fusion protein, proposed for development as the new plague vaccine antigen, the purified protein was observed to form soluble aggregates under certain conditions. Dr. Powell and colleagues used the DAWN EOS and Optilab to characterize the molecular structures of pure F1-V and its constituent subunits, the Yersinia pestis F1 and V proteins. Their investigation showed that aggregation was caused by the F1 subcomponent which forms soluble aggregates 10-times larger than the fusion protein under physiological temperature and salt. SEC-MALS studies also showed that the F1-V fusion protein structure is more stable to cold temperature, high salt, or reducing conditions than the individual subcomponent proteins.

  1. Structure of nerve growth factor complexed with the shared neurotrophin receptor

        X.-L. He, K. C. Garcia, "Structure of nerve growth factor complexed with the shared neurotrophin receptor p75," Science 304 870-875 (2004).

 

Summary: The Garcia team at Stanford University put their DAWN instrument to work in order to help confirm that NGF homodimers and other NT dimers bind to only one neurotrophin receptor p75 in solution. In order to ensure that the 2:1 NGF/p75 stoichiometry isn't an artifact of crystallization, they measured the ligand:receptor stoichiometry and the assembly thermodynamics of p75 complexes in solution with NGF, NT-3, and NT-4/5. MALS and SEC of p75 alone revealed it to be a mixture of a higher and lower molar mass species determined dimer and monomer of 55.1 and 27.0 kDa respectively..

 

  1. Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes

   J. H. Eike, A. F. Palmer, "Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes," Biotechnol. Prog. 20 953-962 (2004)

 

Summary: Professor Palmer's team at the University of Notre Dame used their DAWN, Optilab and Eclipse systems to to measure the absolute molecular weight distribution of PolyHb dispersions in their study of oxidized mono/di/tri/poly saccharides as potential hemoglobin cross-linkers in order to produce oxygen carriers with high oxygen affinities and high molar masses.

 

  1. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein

     R. Ando, H. Hama, M. Yamamoto-Hino, H. Mizuno, A. Miyawaki, "An optical marker based on the 

UV-induced green-to-red photoconversion of a fluorescent protein," PNAS 99(20) 12651-12656 (2002)

 

Summary: The RIKEN group has cloned a fluorescent protein which emits green, yellow and red light, which they have named Kaede. The protein includes a tripeptide, His-Tyr-Gly. Various techniques used to test the transition from one color to another.The team used their DAWN instrument in conjunction with a Shodex HPLC system in order to characterize their Kaede protein. C-terminal His-tagged protein was found to have a mass of 115.0 kDa which is 4.33 times larger than that deduced from the primary structure of the protein. It was therefore concluded that Kaede forms a homotetrameric complex.

 

  1. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

    M. G. Bertero, R. A. Rothery, M. Palak, C. Hou, D. Lim, F. Blasco, J. H. Weiner, N. C. J. Strynadka, "Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A," Nature Structural Biology 10 681-687 (2003).

 

Summary: The team at the University of British Columbia has provided fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop by studying the crystal structure of NarGHI at a resolution of 1.9 Angstroms. The group used their miniDAWN and Optilab DSP instruments to analyze the oligomerization of NarGHI in the presence of 0.7 mM Thesit.

  1. Rational design of low-molecular weight heparins with improved in vivo activity

    M. Sundaram, Y. Qi, Z. Shriver, D. Liu, G. Zhao, G. Venkataraman, R. Langer, R. Sasisekharan, "Rational design of low-molecular weight heparins with improved in vivo activity," PNAS 100(2) 651-656 (2003)

 

Summary: Robert Langer's group at MIT demonstrates a practical analytical method enabling measurement of a structural correlate to in vivo anticoagulant function.They have used this information to to develop low-molecular weight heparins (LMWHs) with increased anticoagulant activity and decreased polydispersity. Desirable in vivo pharmacokinetic properties and the ability to cause release of tissue factor pathway inhibitor from the endothelium are among several beneficial aspects of these LMWHs. The findings demonstrate a simple approach for the creation of designer LMWHs. The group used their miniDAWN in conjunction with a Shodex HPLC system to determine the molar mass and polydispersity of their heparins.

 

  1. Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles

    D. Ishii, K. Kinbara, Y. Ishida, N. Ishii, M. Okochi, M. Yohda, T. Aida, "Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles," Nature 423 628-632 (2003).

 

Summary: The team at the University of Tokyo used their DAWN detector in conjunction with their Shodex HPLC system to find the molar mass of their T.th cpn/CdS nanoparticle inclusion complex. These results demostrate that T.th cpn within the protein-nanoparticle complex preserves its own structural identity, without formation of higher aggregates or dissociation into protein subunts. They further report that GroEL and T.th cpn can also enfold CdS semiconductor nanopartilces, giving them high thermal and chemical stability in aqueous media. Such biological mechanisms integrated into materials science may open a door to conceptually new bioresponsive devices. For information on specifics on their MALS data, please see the Supplementary Information for this publication.

 

  1. Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp 130 complex

    M. J. Boulanger, D.Chow, E. E. Brevnova, K. C. Garcia, "Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp 130 complex," Science 300 2101-2104 (2003).

 

Summary: Professor Garcia's group at Stanford characterized the structure and assembly of the immunoregulatory cytokine, interleukin-6 (IL-6), which activates a cell-surface signalling assembly. A structural model of the complex is presented. The complex forms a hexamer containing 2 IL-6, 2 IL6R-alpha and two gp 130 which assemble sequentially and cooperatively. This structure reveals a conserved architectural blueprint for assembly of all gp130-cytokine signalling complexes. For details on how the DAWN EOS was used, please see the Supporting Online Material for this paper.

 

  1. Crystal structure of a tetradecameric assembly of the association domain of Ca2+/Calmodulin-dependent kinase II

     A. Hoelz, A. C. Narin, J. Kuriyan, "Crystal strucutre of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II," Molecular Cell 11 1241-1251 (2003).

 

Summary: The National Academy of Sciences team led by Professor Kuriyan used their DAWN EOS and Optilab instruments to confirm the crystal structure of the 143 residue association domain of Ca2+/Calmodulin-dependent kinase II (CaMKII). This association domain forms a hub-like assembly which is held together by extensive interfaces. The group has determined an atomic model for the hub and spoke architecture of the association domain, possibly the most complex of the hundreds of pritein kinases in animal cells. This architecture is a critical aspect of CaMKII's to respond to calcium signals and retain a molecular memory of activation events.

 

  1. An "endless" route to cyclic polymers

       C. W. Bielawski, D. Benitez, R. H. Grubbs, "An 'endless' route to cyclic polymers," Science 297 2041-2044 (2002),  California Institute of Technology>

 

Summary: The team lead by Professor Robert Grubbs at Cal Tech developed a novel synthetic route in which the ends of growing polymer chains remain attached to a metal complex throughout the entire polymerization process, which eliminates the need for linear polymeric precursors and high dilution. A GPC system consisting DAWN EOS and Optilab detectors played crucial roles in providing strong physical evidence for circularity of the polymers synthesized. The DAWN EOS detector was used not only for molar mass determination, but also to found that the ratio of mean square radii of cyclic and linear polymers to be approximay 0.5 over a wide range of molar masses, as predicted by theory.

 

  1. Structural evidence for feedback activation by Ras-GTP of the Ras-specific   nucleotide exchange factor SOS

      S. M. Margarit, H. Sondermann, B. E. Hall, B. Nagar, A. Hoelz, M. Pirruccello, D. Bar-Sagi, J. Kuriyan, "Structural evidence for feedback activation by Ras-GTP of the Ras-specific nucleotide exchange factor SOS," Cell 112 685-695 (2003).

 

Summary: This National Academy of Sciences team led by Professor Kuriyan discovered a highly conserved Ras binding site on SOS. It is also shown that Ras-GTP forms ternary complexes with SOScat in solution. A DAWN EOS and  an Optilab were used extensively to characterize their purified SOS complexes as well as verify the theoretically calculated values. Their research indicates the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

 

  1. Light Scattering to Detect Compound Aggregation in Screening Assays

      Dr. Bingyi Yao, Bob Collins, Dr. Michelle Chen "Light scattering to detect compound aggregation in screening assays," DPI 4,1 +32-2-240-26-11

 

Summary: During the high-throughput screening of compound libraries, false positive results can be generated because some compounds form aggregates that nonspecifically inhibit receptors. These compound aggregates are readily detectable by optical methods such as light scattering. This article describes a system that measures dynamic light scattering, using the same conditions as for HTS, allowing the identification of false positives very early in the screening process.

 

  1. Characterization of Hyaluronic Acid with On-Line Differential Viscometry, Multiangle Light Scattering, and Differential Refractometry

    Jason Waters, Danielle Leiske, "Characterization of Hyaluronic Acid with On-Line Differential Viscometry, Multiangle Light Scattering, and Differential Refractometry," LCGC 23,3 (732) 225-9500.

 

Summary: Wyatt Technology in collaboration with Professor Skip Rochefort's group at Oregon State University used a DAWN EOS, an Optilab rEX and a ViscoStar to measure the intrinsic viscosity and Mark-Houwink-Sakurada (MHS) behavior of Hyaluronic Acid (HA). Unusual MHS behavior was demonstrated, perhaps helping explain the large variation in HA MHS coeffiecents that have been reported in the literature.

 

 

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