两种灵芝子实体总蛋白的提取和对比
实验目的:对两种灵芝的子实体中总蛋白进行提取,进行比对。熟悉掌握蛋白含量的测定,总蛋白的提取,双向电泳的原理。
实验原理:蛋白质是含氮的有机化合物。食品与硫酸和硫酸铜、硫酸钾一同加热消化,使蛋白质分解,分解的氨与硫酸结合生成硫酸按。然后碱化蒸馏使氨游离,用硼酸吸收后以硫酸或盐酸标准滴定溶液滴定,根据酸的消耗量乘以换算系数,即为蛋白质的含量。 利用双向电泳将两种灵芝的蛋白分离,并进行对比分析。 实验步骤:
1. 凯氏定氮法测定粗蛋白含量
1.1取20mL灵芝蛋白液加20mL浓硫酸、6g硫酸钾和0.2g硫酸铜消化,将消化液定容到50mL,然后取10mL消化液用自动凯氏定氮仪蒸馏,用10mL 20g/L的硼酸接受氨气,用0.02711 mol/L HCl滴定。 K=(V1-V2)c0.0140
V10
50F100
式中:
X——试样中蛋白质的含量,单位为克每百毫升(g/100 mL); V1——试样消耗盐酸标准滴定液的体积,单位为毫升(mL); V2——试剂空白消耗盐酸标准滴定液的体积,单位为毫升(mL); c——盐酸标准滴定液的浓度,单位为摩尔每升(mol/L);0.0140——1.0 mL盐酸[c(HCl)=1.000 mol/L]标准滴定溶液相当的氮的质量,单位为(g);
v——试样的体积,单位为毫升(mL);
F——氮换算为蛋白质的系数,本实验取6.25。
2双向电泳法对两种灵芝蛋白的分离
2.1总蛋白质的制备
称取0.5g灵芝子实体,用液氮研磨成粉末后加入3ml抽提液(0.1M Tris-HCL(PH8.8),50mM DTT,10mM EDTA)。超声后在4"C、10000g离心10min。上清按1:1体积比加入Tris-饱和酚,室温混悬20—30min后4℃、10000g离心10min,保留酚相。在酚相中加入
l00mMTris-HCL(ph8.8)及10mMDTT后加入4倍体积甲醇(含10mM乙酸铵及10mMDTT),-80℃沉淀2—3h,4℃、10000g离心10min。沉淀用预冷的90%乙醇(含10mMDTT)洗涤2、3次,4"C、10000g离心10min。蛋白溶解在溶解液(8.5M urea,2.5M thiourea,5%CHAPS,
100mMDTT,1%IPGBuffer)中,室温混悬1h,4℃、15000g离心l0min,上清即为蛋白质样本溶液。
2.2蛋自质含量的测定
参照Bradford(1976)[5][11]方法,建立标准曲线和测定样品的蛋白质浓度。以100u1,0.15mol/L的NaCl和lml考马斯亮蓝G-250溶液(10mg考马斯亮蓝G-250溶于5ml的95%的乙醇中,与10ml 85%磷酸混合定容到100ml,滤纸过滤)为空白对照,设牛血清白蛋白(BSA)5个浓度梯度,分别为0.01,0.03,0.05,0.07,0.09(mg/m1),用DU730核酸/蛋白分析仪测定595nm吸光值来绘制标准曲线。每个样品重复测定三次吸光值,取样品的平均吸光值,根据标准益线算出样品浓度。
2.3双向电泳实验
IPGphor第一向等电聚焦电泳主要是依据GE公司的《双向电泳原理与方法中文手册新版》进行。
2.3.1水化上样及等电聚焦
按Bradford法测的蛋白浓度加入120ul蛋白样品(356ug),2ulDTT和78ul水化液至总体积为200ul。混匀,在泡胀盘的持胶槽中加入样品。从阳极端(尖端)开始揭去RR保护膜(撕开后会卷起来的那一面),胶面朝下放入泡胀盘的持胶槽,使之接触样品液面,加入矿物油覆盖整个胶条,水化过夜。将泡胀盘中的胶条转移到滤纸上,胶面朝上.在聚焦盘上加覆盖油.将胶条转移到聚焦盘中加了覆盖油的槽,胶面朝上.将滤纸垫片润湿,放在胶条的两端,滤纸有一半的长度压在胶上.将电极压在滤纸电片上,关好电极.开电源,设置等电聚焦程序并启动。 表1等电聚焦程序
U(V) Time or Vh(Vh)
Stepl 500 500
Grad2 1000 800
Grad3 8000 11300
Step4 8000 2900
当伏小时数达到15500Vh时终止等电聚焦程序,将IPG胶条放入.20。C冰箱备用。
2.3.2胶条的平衡
准备两管10ral的SDS平衡缓冲液(50mM pH8.8的THs.HCI,6M尿素,30%甘油,2%SDS,痕量溴酚蓝),分别加入100mg DTT和250mg
碘乙酰胺。取出胶条,使边缘贴着滤纸让矿物油尽量被吸去,然后放入含DTT的SDS平衡缓冲液,封住管口,平放于摇床平衡15min。取出胶条,放入含碘乙酰胺的SDS平衡缓冲液,封住管口,平放于摇床再平衡15min。
2.3.3 SDS.PAGE采用Ettan DALTsix垂直电泳系统,胶的浓度为12.5%(26 cIIl X20 cm×l mm),将平衡后的胶条放置于已聚合的聚丙稀酰胺凝胶胶面上,用6%的琼脂糖封顶液封闭排除气泡。进行第2向
SDS-PAGE,以恒定功率方式电泳,20。C循环水冷却,先5W/gel电泳20。30rain,待溴酚蓝前沿进入SDS.PAGE约2cm时将电流加大至17W/gel,直至溴酚蓝前沿距离玻璃板下缘lcm时停止电泳。
2.3.4染色(胶体考染法)
用50%的甲醇,10%的乙酸固定10min,再用双蒸水简单的清洗3次,用双蒸水漂洗5min,加染色液(509硫酸铵,0.59考马斯亮蓝,5.88ml 85%磷酸加水至500ml,再加入125ml甲醇),染色时放入密闭的容器中并将其放在摇床上摇,染色时间大于15h,用双蒸水漂洗,直到蛋白点清晰为止。
2.3.5二维凝胶电泳分析
脱色后的双向电泳凝胶用扫描仪ImageScanner扫描,再用图像分析软件ImageMaster 2D Platinum6 0对图谱进行分析,
Experimental principle: The method consists of heating a substance with sulfuric acid, which decomposes the organic substance by oxidation to
liberate the reduced nitrogen as ammonium sulfate. In this step potassium sulfate is added to increase the boiling point of the medium (from 337°F to 373°F / 169°C to 189°C). Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark).
The solution is then distilled with sodium hydroxide (added in small quantities) which converts the ammonium salt to ammonia. The amount of ammonia present (hence the amount of nitrogen present in the sample) is determined by back titration. The end of the condenser is dipped into a solution of boric acid. The ammonia reacts with the acid and the remainder of the acid is then titrated with a sodium carbonate solution with a methyl orange pH indicator.
Degradation: Sample + H2SO4 → (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g)
Liberation of ammonia: (NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) + 2NH3(g)
Capture of ammonia: B(OH)3 + H2O + NH3 → NH4+ + B(OH)4– Back-titration: B(OH)3 + H2O + Na2CO3 → NaHCO3(aq) +
NaB(OH)4(aq) + CO2(g) + H2O
Dimensional (2D) electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. The advantage of 2D protein electrophoresis is it combines two methods of separation and allows the resolution of up to several thousand protein at a time. The first dimension of a 2D gel separates proteins according to their charge using an immobilized pH gradient (IPG) strip. Proteins migrate in the strip until their charge
becomes neutral and then stop their migration. The second dimension gel is typically an SDS gel which separates proteins based on their mass. The results are individual proteins resolved into small circular regions or spots. These gels are often used to analyze samples in proteomics research since the individual spot can be excised, digested and used for mass
spectrometry.
P
p195
两种灵芝子实体总蛋白的提取和对比
实验目的:对两种灵芝的子实体中总蛋白进行提取,进行比对。熟悉掌握蛋白含量的测定,总蛋白的提取,双向电泳的原理。
实验原理:蛋白质是含氮的有机化合物。食品与硫酸和硫酸铜、硫酸钾一同加热消化,使蛋白质分解,分解的氨与硫酸结合生成硫酸按。然后碱化蒸馏使氨游离,用硼酸吸收后以硫酸或盐酸标准滴定溶液滴定,根据酸的消耗量乘以换算系数,即为蛋白质的含量。 利用双向电泳将两种灵芝的蛋白分离,并进行对比分析。 实验步骤:
1. 凯氏定氮法测定粗蛋白含量
1.1取20mL灵芝蛋白液加20mL浓硫酸、6g硫酸钾和0.2g硫酸铜消化,将消化液定容到50mL,然后取10mL消化液用自动凯氏定氮仪蒸馏,用10mL 20g/L的硼酸接受氨气,用0.02711 mol/L HCl滴定。 K=(V1-V2)c0.0140
V10
50F100
式中:
X——试样中蛋白质的含量,单位为克每百毫升(g/100 mL); V1——试样消耗盐酸标准滴定液的体积,单位为毫升(mL); V2——试剂空白消耗盐酸标准滴定液的体积,单位为毫升(mL); c——盐酸标准滴定液的浓度,单位为摩尔每升(mol/L);0.0140——1.0 mL盐酸[c(HCl)=1.000 mol/L]标准滴定溶液相当的氮的质量,单位为(g);
v——试样的体积,单位为毫升(mL);
F——氮换算为蛋白质的系数,本实验取6.25。
2双向电泳法对两种灵芝蛋白的分离
2.1总蛋白质的制备
称取0.5g灵芝子实体,用液氮研磨成粉末后加入3ml抽提液(0.1M Tris-HCL(PH8.8),50mM DTT,10mM EDTA)。超声后在4"C、10000g离心10min。上清按1:1体积比加入Tris-饱和酚,室温混悬20—30min后4℃、10000g离心10min,保留酚相。在酚相中加入
l00mMTris-HCL(ph8.8)及10mMDTT后加入4倍体积甲醇(含10mM乙酸铵及10mMDTT),-80℃沉淀2—3h,4℃、10000g离心10min。沉淀用预冷的90%乙醇(含10mMDTT)洗涤2、3次,4"C、10000g离心10min。蛋白溶解在溶解液(8.5M urea,2.5M thiourea,5%CHAPS,
100mMDTT,1%IPGBuffer)中,室温混悬1h,4℃、15000g离心l0min,上清即为蛋白质样本溶液。
2.2蛋自质含量的测定
参照Bradford(1976)[5][11]方法,建立标准曲线和测定样品的蛋白质浓度。以100u1,0.15mol/L的NaCl和lml考马斯亮蓝G-250溶液(10mg考马斯亮蓝G-250溶于5ml的95%的乙醇中,与10ml 85%磷酸混合定容到100ml,滤纸过滤)为空白对照,设牛血清白蛋白(BSA)5个浓度梯度,分别为0.01,0.03,0.05,0.07,0.09(mg/m1),用DU730核酸/蛋白分析仪测定595nm吸光值来绘制标准曲线。每个样品重复测定三次吸光值,取样品的平均吸光值,根据标准益线算出样品浓度。
2.3双向电泳实验
IPGphor第一向等电聚焦电泳主要是依据GE公司的《双向电泳原理与方法中文手册新版》进行。
2.3.1水化上样及等电聚焦
按Bradford法测的蛋白浓度加入120ul蛋白样品(356ug),2ulDTT和78ul水化液至总体积为200ul。混匀,在泡胀盘的持胶槽中加入样品。从阳极端(尖端)开始揭去RR保护膜(撕开后会卷起来的那一面),胶面朝下放入泡胀盘的持胶槽,使之接触样品液面,加入矿物油覆盖整个胶条,水化过夜。将泡胀盘中的胶条转移到滤纸上,胶面朝上.在聚焦盘上加覆盖油.将胶条转移到聚焦盘中加了覆盖油的槽,胶面朝上.将滤纸垫片润湿,放在胶条的两端,滤纸有一半的长度压在胶上.将电极压在滤纸电片上,关好电极.开电源,设置等电聚焦程序并启动。 表1等电聚焦程序
U(V) Time or Vh(Vh)
Stepl 500 500
Grad2 1000 800
Grad3 8000 11300
Step4 8000 2900
当伏小时数达到15500Vh时终止等电聚焦程序,将IPG胶条放入.20。C冰箱备用。
2.3.2胶条的平衡
准备两管10ral的SDS平衡缓冲液(50mM pH8.8的THs.HCI,6M尿素,30%甘油,2%SDS,痕量溴酚蓝),分别加入100mg DTT和250mg
碘乙酰胺。取出胶条,使边缘贴着滤纸让矿物油尽量被吸去,然后放入含DTT的SDS平衡缓冲液,封住管口,平放于摇床平衡15min。取出胶条,放入含碘乙酰胺的SDS平衡缓冲液,封住管口,平放于摇床再平衡15min。
2.3.3 SDS.PAGE采用Ettan DALTsix垂直电泳系统,胶的浓度为12.5%(26 cIIl X20 cm×l mm),将平衡后的胶条放置于已聚合的聚丙稀酰胺凝胶胶面上,用6%的琼脂糖封顶液封闭排除气泡。进行第2向
SDS-PAGE,以恒定功率方式电泳,20。C循环水冷却,先5W/gel电泳20。30rain,待溴酚蓝前沿进入SDS.PAGE约2cm时将电流加大至17W/gel,直至溴酚蓝前沿距离玻璃板下缘lcm时停止电泳。
2.3.4染色(胶体考染法)
用50%的甲醇,10%的乙酸固定10min,再用双蒸水简单的清洗3次,用双蒸水漂洗5min,加染色液(509硫酸铵,0.59考马斯亮蓝,5.88ml 85%磷酸加水至500ml,再加入125ml甲醇),染色时放入密闭的容器中并将其放在摇床上摇,染色时间大于15h,用双蒸水漂洗,直到蛋白点清晰为止。
2.3.5二维凝胶电泳分析
脱色后的双向电泳凝胶用扫描仪ImageScanner扫描,再用图像分析软件ImageMaster 2D Platinum6 0对图谱进行分析,
Experimental principle: The method consists of heating a substance with sulfuric acid, which decomposes the organic substance by oxidation to
liberate the reduced nitrogen as ammonium sulfate. In this step potassium sulfate is added to increase the boiling point of the medium (from 337°F to 373°F / 169°C to 189°C). Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark).
The solution is then distilled with sodium hydroxide (added in small quantities) which converts the ammonium salt to ammonia. The amount of ammonia present (hence the amount of nitrogen present in the sample) is determined by back titration. The end of the condenser is dipped into a solution of boric acid. The ammonia reacts with the acid and the remainder of the acid is then titrated with a sodium carbonate solution with a methyl orange pH indicator.
Degradation: Sample + H2SO4 → (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g)
Liberation of ammonia: (NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) + 2NH3(g)
Capture of ammonia: B(OH)3 + H2O + NH3 → NH4+ + B(OH)4– Back-titration: B(OH)3 + H2O + Na2CO3 → NaHCO3(aq) +
NaB(OH)4(aq) + CO2(g) + H2O
Dimensional (2D) electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. The advantage of 2D protein electrophoresis is it combines two methods of separation and allows the resolution of up to several thousand protein at a time. The first dimension of a 2D gel separates proteins according to their charge using an immobilized pH gradient (IPG) strip. Proteins migrate in the strip until their charge
becomes neutral and then stop their migration. The second dimension gel is typically an SDS gel which separates proteins based on their mass. The results are individual proteins resolved into small circular regions or spots. These gels are often used to analyze samples in proteomics research since the individual spot can be excised, digested and used for mass
spectrometry.
P
p195