Microfluidic Western blotting of low-molecular-mass proteins.
Microfluidic Western blotting of low-molecular-mass proteins.
复制标题
DOI：
10.1021/ac5024588
复制
发表时间：
2014-11-04
影响因子：
7.4
通讯作者：
Herr, Amy E.
中科院分区：
文献类型：
--
作者：
Gerver, Rachel E.;Herr, Amy E.
我们描述了一种使用Tris tricine不连续缓冲液系统的微流控蛋白质印迹分析（μWestern），适用于宽分子量范围（6.5-116 kDa）的分析。Tris tricine μWestern在封闭的直玻璃微流体通道中完成，该通道容纳了包含光敏二苯甲酮甲基丙烯酰胺单体的双图案聚丙烯酰胺凝胶。在短暂的紫外线（UV）曝光后，水凝胶从用于基于尺寸的分离的分子筛切换到用于原位抗体探测的共价固定支架。电泳控制所有检测阶段，提供纯电子操作，无需泵或阀门进行流体控制。将抗体电泳引入分子筛凝胶中并沿着分子筛凝胶电泳，需要探针必须穿过（i）用于实现高性能分离的瞬时等速电泳中心的不连续凝胶界面和（ii）分离凝胶的整个轴向长度。小分子量物质的通道内抗体探测尤其具有挑战性，因为凝胶必须有效地筛选小蛋白质，同时允许用大分子量抗体进行有效探测。要创建一个良好的控制凝胶界面，我们介绍了一种制造方法，依赖于缓冲液和聚合物前体溶液之间的流体静压力不匹配，以消除当聚合聚合物邻接非聚合聚合物溶液时出现的界面孔径控制问题。与新的扫描抗体探针塞递送方案相结合，与Tris甘氨酸系统相比，Tris tricine μWestern blot的分离分辨率高出40%，蛋白质的去堆积低至6.5 kDa，小孔凝胶的信噪比（SNR）高出100倍，扩大了适用的生物靶标范围。
We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.
登录
查看更多内容
影响因子：
11.2
作者：
DALMAU, J;FURNEAUX, HM;POSNER, JB
通讯作者：
POSNER, JB
影响因子：
7.4
作者：
BURGI, DS;CHIEN, RL
通讯作者：
CHIEN, RL
影响因子：
2.8
作者：
BELANGER, A;VANHALBEEK, H;LABRIE, F
通讯作者：
LABRIE, F
影响因子：
4.2
作者：
Hou, Chenlu;Herr, Amy E.
通讯作者：
Herr, Amy E.
DOI：
10.1016/j.vascn.2009.12.001
发表时间：
2010-03-01
影响因子：
1.9
作者：
MacPhee, Daniel J.
通讯作者：
MacPhee, Daniel J.