Extraction and Analysis of Bacterial Teichoic Acids
Extraction and Analysis of Bacterial Teichoic Acids
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DOI：
10.21769/bioprotoc.3078
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发表时间：
2018-11-05
期刊：
影响因子：
0.8
通讯作者：
Meredith, Timothy C.
中科院分区：
文献类型：
--
作者：
Kho, Kelvin;Meredith, Timothy C.
铁壁酸（TA）是由多元醇磷酸重复单元组成的阴离子聚合物，在革兰氏阳性细菌的细胞壁中含量丰富。壁磷壁酸（WTA）和脂磷壁酸（LTA）在调节细胞壁重塑和产生抗生素耐药性中起重要作用。为了分析TA，我们描述了一种用于WTA和LTA的聚丙烯酰胺凝胶电泳（PAGE）方法。为了提取粗WTA，首先分离肽聚糖小囊，然后水解释放WTA。LTA用1-丁醇提取，脂肪酶预处理，防止聚集，提高PAGE单波段分辨率。这两种ta的粗提取物然后进行PAGE，然后进行阿利新蓝和银染色。这些方案很容易被对快速分析TAs感兴趣的实验室采用，并可用于确定相对丰度，相对聚合物长度和TAs是否糖基化。通过核磁共振（NMR）和电喷雾电离质谱（ESI-MS）分析，可以获得更详细的TA结构和成分信息。
Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide gel electrophoresis (PAGE) method for both WTA and LTA. To extract crude WTA, the peptidoglycan sacculus is first isolated and WTA is then liberated by hydrolysis. LTA is extracted by 1-butanol and pre-treated with lipase to prevent aggregation and improve single-band resolution by PAGE. Crude extracts of both TAs are then subjected to PAGE followed by Alcian blue and silver staining. These protocols are easily adoptable by laboratories interested in rapidly analyzing TAs and can be used determine the relative abundance, relative polymer length and whether TAs are glycosylated. More detailed TA structural and compositional information can be obtained using the described purification protocols by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) analysis.