「常用蛋白质分子量标准参照物」.doc

十、常用蛋白质分子量标准参照物 高分子量标准参照 中分子量标准参物 低分子量标准参照蛋白质Mr蛋白质Mr蛋白质Mr肌球蛋白半乳糖苷酶磷酸化酶B牛血清清蛋白过氧化氢酶醛缩酶212 000116 00097 40066 20057 00040 000磷酸化酶B牛血清清蛋白谷氨酸脱氢酶卵清蛋白醛缩酶碳酸酐酶大豆胰蛋白酶抑制剂溶菌酶97 40066 20055 00042 70040 00031 00021 50014 400碳酸酐酶大豆胰蛋白酶制剂马心肌球蛋白溶菌酶肌球蛋白（F1）肌球蛋白（F2）肌球蛋白（F3）31 00021 50016 90014 4008 1006 2002 500十一、层析法常用数据表及性质（一） 离子交换纤维素目前常用的离子交换纤维素列于下表DEAE纤维素形状长度（微米）交换当量（毫克当量/克）蛋白吸附容量 （毫克/克）床体积（毫升/克）胰岛素（pH8.5）牛血清清蛋白（pH8.5）pH6.0pH7.5DE-22DE-23DE-32DE-52CM-纤维素CM-22CM-23CM-32CM-52改良纤维性 *同上（除细粒）微粒性（干粉）同上（溶胀）改良纤维性同上（除细粒）微粒性（干粉）同上（溶胀）1240018400246324631240018400246324631.00.11.00.11.00.11.00.10.60.060.60.061.00.11.00.1750750850850溶菌酶(pH5.0)6006001,2601,2604504506606607S球蛋白（pH5.0）1501504004007.78.36.06.0pH5.07.79.16.86.87.79.16.36.3pH7.57.79.16.76.7*：英国Whatman厂的型号，原来有旧型号如DE-1，为长纤维性，长度1000微米。还有DE-11，纤维性，50250微米，对牛血清清蛋白的吸附容量仅为130毫克/克。（二）聚丙烯酰胺凝胶的技术数据型号排阻的下限（Mr）分级分离的范围（Mr）膨胀后的床体积（mL/g干凝胶）膨胀所需最少时间（室温，小时）Bio-gel-P-2Bio-gel-P-4Bio-gel-P-6Bio-gel-P-10Bio-gel-P-30Bio-gel-P-60Bio-gel-P-100Bio-gel-P-150Bio-gel-P-200Bio-gel-P-3001 6003 6004 60010 00030 00060 000100 000150 000200 000300 0002002 0005004 0001 0005 0005 00017 00020 00050 00030 00070 00040 000100 00050 000150 00080 000300 000100 000400 0003.85.88.812.414.919.019.024.034.040.0242424241012101224244848注：上述各种型号的凝胶都是亲水性的多孔颗粒，在水和缓冲溶液中很容易膨胀，生产厂为Bio-Rad Laboratories,Rich-mond, California, U. S. A.（三）琼脂糖凝胶的技术数据（琼脂糖是琼脂内非离子型的组分，它在0 4，pH 49范围内是稳定的。）名称、型号凝胶内琼脂糖百分含量（W/W）排阻的下限（Mr）分级分离的范围（Mr）生产厂商Sagavac 10Sagavac 8Sagavac6Sagavac4Sagavac21086422.5105710521061510615010611042.51052.5104710551042106210515106510515107Seravac Laboratories,Maidenhead, EnglandBio-GelA-0.5MBio-GelA-1.5MBio-GelA-5MBio-GelA-15MBio-GelA-50MBio-GelA-150M10864210.51051.51065106151055010615010611042.510611041.5106110451064104151061105501061106150106Bio-Rad Laboratories,California,U.S.A.（四）凝胶过滤层析介质的技术数据凝胶过滤介质名称分离范围颗粒大小（m）特性/应用pH稳定性工作耐压（Mpa）最 快流 速（cm/h）Superdex 30 Superdex 75 Superdex200 Superose 6 Superose 12 SephacrylS-100 HRSephacrylS-200 HRSephacrylS-300 HRSephacrylS-400 HRSepharose 6Fast FlowSepharose 4Fast FlowSepharose 2BSepharose 4BSepharose 6BSepharoseCL-2BSepharoseCL-4BSepharoseCL-6B100003000700003 000 100006000005000510610003000001000 1000005000250000100001.510620000810610000410660000201067000040106600002010610000410670000401066000020105100004106244424442444204020402575257525752575平均90平均90602004516545165602004516545165肽类、寡糖、小蛋白等重组蛋白、细胞色素单抗、大蛋白蛋白、肽类、多糖、核酸蛋白、肽类、寡糖、多糖肽类、小蛋白蛋白，如清蛋白蛋白、抗体多糖、具延伸结构的大分子如蛋白多糖、脂质体巨大分子巨大分子如重组乙型肝炎表面抗原蛋白、大分子复合物、病毒、不对称分子如核酸和多糖（蛋白多糖）蛋白、多糖蛋白、多糖蛋白、大分子复合物、病毒、不对称分子如核酸和多糖（蛋白多糖）蛋白、多糖蛋白、多糖312312312312312311311311 3112122124949493133130.30.30.30.40.70.20.20.2 0.20.10.10.0040.0080.020.0050.0120.021001001003030203920392039 20393002501011.514152630凝胶过滤介质名称分离范围颗粒大小（m）特性/应用pH稳定性工作干凝胶溶胀体积（mL/g）溶胀最少平衡时间（h）最快流速(cm/h)室温沸水SephadexG-10SephadexG-15SephadexG-25 CoarseSephadexG-25MediumSephadexG-25 FineSephadexG-25 SuperfineSephadexG-50 CoarseSephadexG-50 MediumSephadexG-50 FineSephadexG-50 SuperfineSephadexG-75SephadexG-75 SuperfineSephadexG-100SephadexG-100SuperfineSephadexG-150SephadexG-150SuperfineSephadexG-200SephadexG-200Superfine700200000）,在工业传统应用上具有重要作用2100.11475QAE SephadexA-501.2mg甲状腺球蛋白80mgHSA干粉40120批量生产和预处理用，分离中等大小的生物分子（30200000）211 0.0145SP SephadexC-251.1mg IgG70mg牛羰合血红蛋白230mg核糖核酸酶干粉40120纯化低分子量蛋白、多肽、核苷以及巨大分子（Mr200000）,在工业传统应用上具有重要作用2100.13475SP SephadexC-508mg IgG110mg牛羰合血红蛋白干粉40120批量生产和预处理用，分离中等大小的生物分子（30200000）2100.0145DEAE SephadexA-251mg甲状腺球蛋白30mg HAS140mg a-乳清蛋白干粉40120纯化低分子量蛋白、多肽、核苷以及巨大分子（Mr200 000）,在工业传统应用上具有重要作用290.11475DEAE SephadexA-502mg 甲状腺球蛋白110mg HSA干粉40120批量生产和预处理用，分离中等大小的生物分子（Mr200 000），在工作传统应用上具有重要作用290.1145CM SephadexC-251.6mg IgG 70mg牛羰合血红蛋白190mg核糖核酸酶干粉40120纯化低分子量蛋白、多肽、核苷以及巨大分子（Mr200 000），在工业传统应用上具有重要作用6130.13475CM SephadexC-507mg IgG140mg牛羰合血红蛋白120mg核糖核酸酶干粉40120批量生产和预处理用，分离中等大小的生物分子（30200000）6100.0145十二、常用缓冲溶液的配制方法1 甘氨酸盐酸缓冲液（0.05mol/L）X毫升0.2 mol/L甘氨酸+Y毫升0.2 mol/L HCI，再加水稀释至200毫升pHXYpHXY2.02.42.62.85050505044.032.424.216.83.03.23.43.65050505011.48.26.45.0甘氨酸分子量 = 75.07，0.2 mol/L甘氨酸溶液含15.01克/升。2邻苯二甲酸盐酸缓冲液（0.05 mol/L）X毫升0.2 mol/L邻苯二甲酸氢钾 + 0.2 mol/L HCl，再加水稀释到20毫升pH(20)XYpH(20)XY2.22.42.62.83.0555554.0703.9603.2952.6422.0223.23.43.63.855551.4700.9900.5970.263邻苯二甲酸氢钾分子量 = 204.23，0.2 mol/L邻苯二甲酸氢溶液含40.85克/升3磷酸氢二钠柠檬酸缓冲液pH0.2mol/L Na2HPO4（毫升）0.1mol/L柠檬酸（毫升）pH0.2mol/L Na2HPO4 （毫升）0.1mol/L柠檬酸（毫升）2.22.42.62.83.03.23.43.63.84.04.24.44.64.85.00.401.242.183.174.114.945.706.447.107.718.288.829.359.8610.3010.6018.7617.8216.8315.8915.0614.3013.5612.9012.2911.7211.1810.6510.149.705.25.45.65.86.06.26.46.66.87.07.27.47.67.88.010.7211.1511.6012.0912.6313.2213.8514.5515.4516.4717.3918.1718.7319.1519.459.288.858.407.917.376.786.155.454.553.532.611.831.270.850.55Na2HPO4分子量 = 14.98，0.2 mol/L溶液为28.40克/升。Na2HPO4-2H2O分子量 = 178.05，0.2 mol/L溶液含35.01克/升。C4H2O7H2O分子量 = 210.14，0.1 mol/L溶液为21.01克/升。4柠檬酸氢氧化钠-盐酸缓冲液pH钠离子浓度（mol/L）柠檬酸（克）C6H8O7H2O氢氧化钠(克)NaOH 97%盐酸（毫升）HCl（浓）最终体积（升）2.23.13.34.35.35.86.50.200.200.200.200.350.450.3821021021021024528526684838383144186156160116106456810512610101010101010使用时可以每升中加入1克克酚，若最后pH值有变化，再用少量50%氢氧化钠溶液或浓盐酸调节，冰箱保存。5柠檬酸柠檬酸钠缓冲液（0.1 mol/L）pH0.1 mol/L柠檬酸（毫升）0.1 mol/L柠檬酸钠（毫升）pH0.1 mol/L柠檬酸（毫升）0.1 mol/L柠檬酸钠（毫升）3.03.23.43.63.84.04.24.44.64.818.617.216.014.914.013.112.311.410.39.21.42.84.05.16.06.97.78.69.710.85.05.25.45.65.86.06.26.46.68.27.36.45.54.73.82.82.01.411.812.713.614.515.316.217.218.018.6柠檬酸C6H8O7H2O：分子量210.14，0.1 mol/L溶液为21.01克/升。柠檬酸钠Na3 C6H5O72H2O：分子量294.12，0.1 mol/L溶液为29.41克/毫升。6乙酸乙酸钠缓冲液（0.2 mol/L）pH(18)0.2 mol/L NaAc(毫升)0.3 mol/L HAc(毫升)pH(18)0.2 mol/LNaAc(毫升)0.3 mol/LHAc(毫升)2.63.84.04.24.44.60.751.201.802.653.704.909.258.808.207.356.305.104.85.05.25.45.65.85.907.007.908.609.109.404.103.002.101.400.900.60Na2Ac3H2O分子量 = 136.09，0.2 mol/L溶液为27.22克/升。7磷酸盐缓冲液（1）磷酸氢二钠磷酸二氢钠缓冲液（0.2）pH0.2 mol/L Na2HPO4(毫升)0.3 mol/L NaH2PO4(毫升)pH0.2 mol/L Na2HPO4(毫升)0.3 mol/L NaH2PO4(毫升)5.85.96.06.16.26.36.46.56.66.76.86.98.010.012.315.018.522.526.531.537.543.549.555.092.090.087.785.081.577.573.568.562.5 56.551.045.07.07.17.27.37.47.57.67.77.87.98.061.067.072.077.081.084.087.089.591.593.094.739.033.028.023.019.016.013.010.58.57.05.3Na2HPO42H2O分子量 = 178.05，0.2 mol/L溶液为85.61克/升。Na2HPO42H2O分子量 = 358.22，0.2 mol/L溶液为71.64克/升。Na2HPO42H2O分子量 = 156.03，0.2 mol/L溶液为31.21克/升。（2）磷酸氢二钠磷酸二氢钾缓冲液（1/15 mol/L）pHmol/L/15Na2HPO4(毫升)mol/L/15KH2PO4(毫升)pHmol/L/15Na2HPO4(毫升)mol/L/15KH2PO4(毫升)4.925.295.916.246.476.646.816.980.100.501.002.003.004.005.006.009.909.509.008.007.006.005.004.007.177.387.738.048.348.678.187.008.009.009.509.759.9010.003.002.001.000.500.250.100Na2HPO42H2O分子量 = 178.05，1/15M溶液为11.876克/升。KH2PO4分子量 = 136.09，1/15M溶液为9.078克/升。8磷酸二氢钾氢氧化钠缓冲液（0.05M）X毫升0.2M K2PO4 + Y毫升0.2N NaOH加水稀释至29毫升 pH(20)X (毫升)Y（毫升）pH(20)X (毫升)Y（毫升）5.86.06.26.46.66.85555550.3720.5700.8601.2601.7802.3657.07.27.47.67.88.05555552.9633.5003.9504.2804.5204.6809巴比妥钠-盐酸缓冲液（18）pH0.04M巴比妥钠溶液0.2V盐酸pH0.04M巴比妥钠溶液(毫升)0.2N盐酸(毫升)6.87.07.27.47.67.88.08.210010010010010010010010018.417.816.715.313.411.479.397.218.48.68.89.09.29.49.61001001001001001001001005.213.822.521.651.130.700.35巴比妥钠盐分子量=206.18;0.04M溶液为8.25克/升10Tris盐酸缓冲液（0.05M，25）50毫升0.1M三羟甲基氨基甲烷（Tris）溶液与X毫升0.1N盐酸混匀后，加水稀释至100毫升。pHX(毫升)pHX(毫升)7.107.207.307.407.507.607.707.807.908.0045.744.743.442.040.338.536.634.532.029.28.108.208.308.408.508.608.708.808.9026.222.919.917.214.712.410.38.57.0三羟甲基氨基甲烷（Tris）HOCH2 CH2OH CHOCH2 NH2分子量=121.14;0.1M溶液为12.114克/升。Tris溶液可从空气中吸收二氧化碳，使用时注意将瓶盖严。11硼酸硼砂缓冲液（0.2M硼酸根）pH0.05M硼砂（毫升）0.2M硼砂（毫升）pH0.05M硼砂（毫升）0.2M硼酸（毫升）7.47.67.88.01.01.52.03.09.08.58.07.08.28.48.79.03.54.56.08.06.55.54.02.0硼砂Na2B4O7H2O,分子量=381.43;0.05M溶液（=0.2M硼酸根）含19.07克/升。硼酸H2BO3,分子量=61.84,0.2M溶液为12.37克/升。硼砂易失去结晶水，必须在带塞的瓶中保存。11硼酸-硼砂缓冲液（0.2M硼酸根）pH0.05M硼砂（毫升）0.2M硼酸（毫升）pH0.05M硼砂（毫升）0.2M硼酸（毫升）7.47.67.88.01.01.52.03.09.08.58.07.08.28.48.79.03.54.56.08.06.55.54.02.0硼砂Na2B4O710H2O,分子量=381.43;0.05M溶液（=0.2M硼酸根）含19.07克/升。硼酸H2BO3，分子量=61.84, 0.2M溶液为12.37克/升。硼砂易失去结晶水，必须在带塞的瓶中保存。12甘氨酸氢氧化钠缓冲液（0.05M）X毫升0.2M甘氨酸+Y毫升0.2NnaOH加水稀释至200毫升pHXYpHXY8.68.89.09.29.450505050504.06.08.812.016.89.69.810.010.410.6505050505022.427.232.038.645.5甘氨酸分子量=75.07;0.2M溶液含15.01克/升。13硼砂-氢氧化钠缓冲液（0.05M硼酸根）X毫升0.05M硼砂+Y毫升0.2NNaOH加水稀释至200毫升pHXYpHXY9.39.49.65050506.011.023.09.810.010.150505034.043.046.0硼砂Na2B4O710H2O,分子量=381.43;0.05M溶液为19.07克/升。14碳酸钠-碳酸氢钠缓冲液（0.1M）Ca2+、Mg2+存在时不得使用pH0.1MNa2CO3(毫升)0.1MN2HCO3(毫升)20379.169.409.519.789.9010.1410.2810.5310.838.779.129.409.509.729.9010.0810.2810.57123456789987654321Na2CO210H2O分子量=286.2;0.1M溶液为28.62克/升。N2HCO3分子量=84.0;0.1M溶液为8.40克/升。15“PBS”缓冲液PH7.67.47.27.0H2ONaClNa2HPO4NaH2PO410008.52.20.110008.52.20.210008.52.20.3100ml8.5g2.2g0.4g十三、实验室中常用酸碱的比重和浓度名称分子式分子量比重面分浓度%（W/W）当量浓度（粗略）N配1升1N溶液所需毫升数盐酸HCl36.471.1937.212.0841.1835.411.81.1020.06.0硫酸H2SO498.091.8495.636.0281.1824.86.0硝酸HNO263.021.4270.9816.0631.4065.314.51.2032.366.1冰乙酸CH3COOH60.051.0599.517.459乙酸CH3COOH98.06366.0磷酸H2PO435.051.7185.015,30,45(依反应而定)67(以15N计)氨水NH4OH0.9015670.90427.014.3700.9125.013.40.9610.05.6氢氧化钠溶液NaOH40.01.550.01953十四、硫酸铵饱和度的常用表1调整硫酸铵溶液饱和度计算表（25）硫整硫酸铵终浓度,%饱和度10202530333540455055606570758090100每1升溶液加固体硫酸铵的克数硫酸铵初浓度，%饱和度05611414417619620924327731335139043047251656166276710578611813715018321625128832636540644949459269420295978911231551902252623003403824245206192530496193125158193230267307348390485583301930629412716219823527331435644954633124374107142177214252292333426522353163941291642002381783194115064031639713216820524528537546945326599134171210250339431503366101137176214302392553367103141179264353603469105143227314653470107190275703572153237753611519880771579079在25下，硫酸铵溶液由初浓度调到终浓度时，每升溶液所加固体硫酸铵的克数。2 调整硫酸铵溶液饱和度计算表（0）在0硫酸铵终浓度，%饱和度20253035404550556065707580859095100每100毫升溶液加固体硫酸铵的克数硫酸铵初浓度，%饱和度010.613.416.419.422.625.829.132.636.139.843.647.651.655.960.365.069.757.910.813.716.619.722.926.229.633.136.840.544.448.452.657.061.566.2105.38.110.913.916.920.023.326.630.133.737.441.245.249.353.658.162.7152.65.48.211.114.117.220.423.727.130.634.338.142.045.050.354.759.22002.75.58.311.314.317.520.724.127.631.234.938.742.746.951.255.72502.75.68.411.514.617.921.124.528.031.735.539.543.647.852.23002.85.68.611.714.818.121.424.928.532.336.240.244.548.83502.85.78.711.815.118.421.825.429.132.936.941.045.34002.95.88.912.015.318.722.225.829.633.537.641.84502.95.99.012.315.519.022.626.330.234.238.35003.06.09.212.515.919.423.026.830.834.85503.16.29.512.916.419.723.527.331.36003.16.39.713.216.820.123.127.96503.16.39.713216.820.524.47003.26.59.913.417.120.97503.26.610.113.717.48003.36.710.313.98503.46.810.59003.47.09503.51000在0下，硫酸铵溶液由初浓度调到终浓度时，每100毫升溶液所加固体硫酸铵的克数。十五、常见蛋白质分子量参考值 （单位：dalton）蛋白质分子量肌球蛋白myosin甲状腺球蛋白thyroglobulin-半乳糖苷酶-galactosidase副肌球蛋白paramyosin磷酸化酶aphosphorylase a血清白蛋白serum albuminL-氨基酸氧化酶L-amino acid oxidase地氧化氢酶catalase丙酮酸激活酶pyruvate kinase谷氨酸脱氢酶glutamate dehydrogenase亮氨酸氨肽酶glutamae dehydrogenase-球蛋白，H链-globulin, H chain延胡索酸酶（反丁烯二酸酶）fumarase卵白蛋白ovalbumin醇脱氢酶（肝）alcohol dehydrogenase (liver)烯醇酶enolase醛缩酶aldolase肌酸激酶creatine kinase胃蛋白酶原pepsinogenD-氨基酸氧化酶D-amino acid oxidase醇脱氢酶（酵母）alcohol dehydroge