第十章蛋白質(zhì)和氨基酸的測(cè)定-文庫(kù)吧
2025-07-17 13:32 本頁(yè)面
【正文】 L 100 ml Methanol 100 ml Glacial acetic acid 800 ml Water 4. Staining Procedure 1. Pick up the gel into (20 ml, usually enough) Staining solution in a container and agitate for 10 min for mm Gel and 20 min and mm gel. The staining solution can be reused several times. 2. Take the gel out and rinse the gel with a few changes of water in a new container 3. Add 50 ml destaining solution. Strong bands are visiable immediately on a light box, and 1 hour usually is enough. 4. To destain pletely, change destaining solution 23 times and agitate overnight. 5. Scan or take photo to record the result. Coomassie Blue STAINING PROCEDURES 4. 銀染法 (Silver staining): can detect as little as 2 ng of protein in a single band in SDSPAGE Gel. Reagents: Silver nitrate Sodilum hydroxide 30% ammonium hydroxide citric acid 38% formaldehyde Methanol Acetic acid Staining procedure 1. Sock gel in 50% Methanol10% acetic acid for at least 1 hour with 23 chan ges of socking solution 2. Rinse gel with 3 changes of water for total 30 minutes. 3. silver staining for 15 mins 4. Rinse gel with twice in deionized water 5. Develop gel with developing solution 6. stop development by ringsing in 1% acetic acid. 7。 wash gel in water for at least 1 hour. 2DE 2DE: the technique to separate proteins in the first dimension according to their isoelectric point, by Isoelectric Focusing(IEF), and in the second dimension according to their molecular weight, by SDSPAGE. 2DE bined with protein identification basing on microsequencing, amino acid position and Mass spectrometry, provides an invaluable tool for proteomic studies. Step 1 — Sample Prep Step 2 — FirstDimension (IEF) Separation Step 3 — SecondDimension (SDSPAGE) Separation Step 4 — Protein Detection by Staining /Destaining 凱氏定氮法 凱氏定氮法是先測(cè)定總氮量 , 然后乘以蛋白質(zhì)換算系數(shù)，即可得到粗蛋白質(zhì)含量。可用于所有動(dòng)、植物食品的蛋白質(zhì)含量測(cè)定。但因樣品中常含有核酸、生物堿、含氮類(lèi)脂、卟啉以及含氮色素等非蛋白質(zhì)的含氮化合物， 故結(jié)果稱(chēng)為粗蛋白質(zhì)含量。 凱氏定氮法由 Kieldahl于 1833年首先提出，經(jīng)過(guò)長(zhǎng)期改進(jìn)，迄今已演變成常量法、微量法、自動(dòng)定氮儀法、半微量法及改良凱氏法等多種，至今仍被作為標(biāo)準(zhǔn)檢驗(yàn)方法。在此主要介紹 常量凱氏定氮法的原理及步驟 。 原理 : 樣品與濃硫酸和催化劑一同加熱 消化 ，使蛋白質(zhì)分解，其中碳和氫被氧化成二氧化碳和水逸出，而部分硫酸被還原成二氧化硫，樣品中的 有機(jī)氮轉(zhuǎn)化為氨 與 過(guò)量的硫酸結(jié)合成硫酸銨 。 然后 加堿蒸餾 ， 使氨蒸出，用弱酸硼酸吸收后再以標(biāo)準(zhǔn)強(qiáng)酸如鹽酸或硫酸溶液滴定 ，根據(jù)標(biāo)準(zhǔn)酸消耗量可計(jì)算出蛋白質(zhì)的含量。 催化劑 煮沸 消化： NCOC + 濃 H2SO4 （ NH4） 2SO4 + CO2 + SO2 + H2O 氨化： 2NaOH +（ NH4） 2SO4 2NH3↑+Na2SO4 + 2H2O 吸收 : 2NH3 + 4H3BO3 （ NH4） 2B4O7 + 5H2O 滴定： （ NH4） 2B4O7 + 5H2O + 2HCl 2NH4Cl + 4H3BO3 (注 : NCOC, Nitrogen containin
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