| Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
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Author:
Date:
2016-04-05
[Abstract] Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).
[摘要] 高质量RNA的提取是定量逆转录PCR（qRT-PCR）和微阵列分析的必要步骤。 然而，从含有大量多糖，脂质和次级代谢物的水果材料中提取高质量RNA是不容易的。 Wan和Wilkins（1994）开发了"热硼酸盐方法"来分离高质量的RNA。 在这里，我们描述了"热硼酸盐方法"的修饰的协议，从葡萄浆果皮肤中分离高质量的RNA用于qRT-PCR和微阵列分析（Suzuki等人，2015a; Suzuki等人 al。，2015b）。
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| Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
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Author:
Date:
2015-10-20
[Abstract] Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods ...
[摘要] 在异质发酵细菌物种，例如otakiensis的乳酸杆菌（Lactobacillus otakiensis）的培养基中观察到D-亮氨酸，D-异亮氨酸 - 异亮氨酸和D-缬氨酸的显着积累。 otakiensis）JCM 15040.从细菌细胞中纯化导致这种积累的消旋酶，异亮氨酸2-差向异构酶。这是首次报道的在乳酸菌中这种D-支链氨基酸的产生的观察结果，以及在任何生物体中具有异亮氨酸2-差向异构酶活性的消旋酶的第一个实例。在所述的方案中，我们介绍从L中纯化该蛋白质的方法。因为没有鉴定到对该酶具有高亲和力的特异性配体，所以使用硫酸铵级分，四种类型的柱层析和制备型Native-PAGE，不使用亲和柱层析进行纯化。我们希望协议将提供有用的信息用于纯化不能容易地使用亲和柱层析纯化的酶。
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