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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >P148-S A Sensitive and Simple Procedure for Staining Proteins on Either Nitrocellulose or PVDF Membranes Based on the Fluorophore Epicocconone
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P148-S A Sensitive and Simple Procedure for Staining Proteins on Either Nitrocellulose or PVDF Membranes Based on the Fluorophore Epicocconone

机译：P148-S是一种敏感且简单的程序可基于荧光团表艾可可酮对硝酸纤维素膜或PVDF膜上的蛋白质进行染色

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Epicocconone, a naturally occurring fluorophore that alters its fluorescent character upon binding to proteins is the basis for a number of new labeling technologies ranging from liquid protein quantitation to gel stains and cell stains. LavaPurple is a highly sensitive total protein gel stain that may be used as a stain for nitrocellulose and PVDF blots. The high sensitivity of the stain, coupled with the unique reversible covalent binding of epicocconone, make it an ideal blot stain as it is easily removed by pH change thus not interfering with downstream immuno-staining, mass spectrometry or Edman sequencing.Aim: This study measures the sensitivity of LavaPurple as a blot stain on both nitrocellulose and PVDF membranes, as well as the compatibility of the stain with immuno-staining.Method: a dilution series of Low molecular weight SDS-PAGE calibration markers (GE Healthcare) were separated by electrophoresis and electro-blotted onto either nitrocellulose or PVDF. The blots were then stained with LavaPurple and image analysis performed to determine sensitivity and linearity of the stain. Cell lysate from ovarian cancer cells and rat fibroblasts were separated by 2D GE and blotted onto both nitrocellulose and PVDF membranes before staining with LavaPurple. The blots were then used for immuno-staining and the efficiency of visualization compared with and without total protein pre-staining.Results: LavaPurple was able to visualise proteins on blots at less than 250pg/band loaded on a 1D gel and did not inhibit subsequent antibody binding to target proteins on either 1D or 2D blots. Furthermore the stain could be easily removed from the blot for subsequent staining with other fluorophores.Conclusions: LavaPurple is a highly sensitive stain that is suitable for total protein visualization on blots either for spot removal or prior to immuno-staining.
机译：Epicocconone是一种天然存在的荧光团，在与蛋白质结合后会改变其荧光特性，这是许多新的标记技术的基础，这些标记技术从液体蛋白质定量到凝胶染色和细胞染色，不一而足。 LavaPurple是一种高度敏感的总蛋白凝胶染料，可用作硝酸纤维素和PVDF印迹的染料。该染色剂的高灵敏度加上表艾康酮的独特可逆共价结合，使其成为理想的印迹染色剂，因为它很容易被pH改变而去除，因此不会干扰下游的免疫染色，质谱或Edman测序。目的：本研究分别测定LavaPurple在硝酸纤维素膜和PVDF膜上作为印迹污渍的敏感性以及该污渍与免疫染色的相容性。方法：用低分子量SDS-PAGE校准标记（GE Healthcare）稀释系列电泳并在硝酸纤维素或PVDF上进行电印迹。然后将印迹用LavaPurple染色，并进行图像分析以确定染色的灵敏度和线性。用2D GE分离来自卵巢癌细胞和大鼠成纤维细胞的细胞裂解液，并在用LavaPurple染色之前将其印迹到硝酸纤维素膜和PVDF膜上。然后将印迹用于免疫染色，并与不进行总蛋白预染色相比，将其进行可视化。结果：LavaPurple能够以小于250pg /条带的浓度在1D凝胶上以低于250pg /条的速度对蛋白质进行可视化，并且不会抑制后续抗体与1D或2D印迹上的靶蛋白结合。此外，可以很容易地从污点上去除污点，以便随后用其他荧光团进行染色。结论：LavaPurple是一种高度敏感的污点，适用于污点上或免疫染色之前的污点上的总蛋白可视化。

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