Basu Koli, Garnham Christopher P, Nishimiya Yoshiyuki, Tsuda Sakae, Braslavsky Ido, Davies Peter
Department of Biomedical and Molecular Sciences, Queen's University.
J Vis Exp. 2014 Jan 15(83):e51185. doi: 10.3791/51185.
Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.
抗冻蛋白（AFPs）在多种耐寒生物中表达，以防止或减缓体内冰晶生长。AFPs通过其冰结合表面与特定的冰面结合。基于荧光的冰面亲和力（FIPA）分析是一种用于确定AFPs结合的冰面的改良技术。FIPA基于最初用于确定AFP结合冰面的冰蚀刻方法。它在缩短的实验时间内产生更清晰的图像。在FIPA分析中，AFPs用嵌合标签或共价染料进行荧光标记，然后缓慢掺入预先形成半球形并定向以确定a轴和c轴的宏观单晶冰中。在紫外线下对结合了AFP的冰半球进行成像，使用滤光片阻挡非特异性光以可视化结合了AFP的平面。AFPs的荧光标记允许实时监测AFP吸附到冰中。已发现这些标签不会影响AFPs结合的平面。FIPA分析还引入了在同一单晶冰上结合不止一种不同标记的AFP的选项，以帮助区分它们的结合平面。FIPA的这些应用有助于推动我们对AFPs如何与冰结合以阻止其生长以及为何许多产生AFP的生物表达多种AFP异构体的理解。
