高级生化技术 2

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Interfering s Substance ubstances
CHAPS
蔗糖sucrose (2氨基 EGTA(乙二醇双 乙二醇双(2(2-氨基 )四乙酸 ) 乙醚 乙醚) 四乙酸) ) EDTA(乙二胺四乙酸 乙二胺四乙酸) 硫酸胺Ammonium sulphate Triton X-100 Dithiothreitol二硫苏糖醇
After phenol reagent is added into the tubes for 30 min, absorbance is read at wavelength 650 nm on condition that blank tube is adjusted to zero.
Lowry Assay ( Folin -Phenol Method ) (Folin Folin-Phenol
紫外分光光度法 UV spectrophotometry
� Absorption of UV (A280) by proteins depends on the Tyrosine and Tryptophan content (and a very small extent on the amount of Phe and disulfide bonds ). A280 is directly bonds). proportional to the protein content. � Advantages: rapid, simple, recoverable. � Disadvantages: less sensitive (0.1～1mg/mL) ； protein-to-protein variation.
Quantitative analysis 1
� draw a standard curve shows how absorbance changes with the concentration of a solution . � According to absorbance of samples, concentrations of serum protein can be looked up in the standard curve.
Step 1
Step 2
Blue compounds ( A650) (A The degree of blue has a direct proportion to content of the prot . prot.
Lowry Assay ( Folin -Phenol Method ) (Folin Folin-Phenol
� C=A/ ε1mol/L , or C=A/ E 1% ε1mol/L represents the the absorbance given by 1cm thick sample of a 1mol/L solution of the substance. E1% represents the the absorbance given by 1cm thick sample of a 1% solution of the substance.
紫外分光光度法 UV spectrophotometry
Quantitative analysis of a pure sample ⑴ Read A280 and A260 of the sample, then use the following empirical formula to estimate protein concentration. Kalckar formula: ① LowryLowry-Kalckar
双缩脲法 Biuret Test � Principle:
The reactivity of the peptide bonds with Cu2+ under alkaline conditions to form purple biuret complex. The most widely used method for protein determinatio
Protein - copper complex formation under alkaline conditions Protein - copper complex reduce phosphomolybdic phosphoyungstic acid to acidacid-phosphoyungstic blue compounds.
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Contents
1源自文库
蛋白质 溶液的 浓度测定 蛋白质溶液的 溶液的浓度测定
Determination of Protein Concentration
2
蛋白质的凝胶电泳分离
Protein gel electrophoresis
Biuret Complexes ( purple color )
The absorbance of the Cu2+-protein complex is measured at 540 nm.
酚试剂法 Folin -Phenol Method / Folin-Phenol Lowry assay
The residues of tyrosine and tryptophan reduce phosphoyungstic acid (磷钨酸—磷钼酸) phosphomolybdic acidacid-phosphoyungstic in phenol reagent to blue compounds.
Advantages
Disadvantages
Procedure
7 test tubes are used and the operation is done according to the following table
Reagents (ml) Blank Protein standard solution µg/ml) (250 (250µ 0.9%NaCl solution Alkaline copper sulfate reagent ----0.5 2.5 1 0.1 0.4 2.5 2 0.2 0.3 2.5 3 0.3 0.2 2.5 4 0.4 0.1 2.5 5 0.5 ----2.5 diluted sample 0.5 ----2.5
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特定蛋白质的鉴定分析
Western Blotting
蛋白质定量常用方法
Methods of Protein Concentration Determination 1. 凯氏微量定氮法 The Kjeldahl method 2. 紫外分光光度法 UV spectrophotometric assay 3. 双缩脲法 Biuret Test 4.酚试剂法 Folin-Phenol Method /Lowry assay 5.考马氏亮蓝-G250 Bradford (Dye-Binding) Assay 6.二喹啉甲酸法 BCA (Bicinchoninic Acid ) Assay
Mix the contents of tubes, incubate 20 min at room temperature
Phenol reagent Equal protein concentration (mg/L) 0.25 0 0.25 50 0.25 100 0.25 150 0.25 200 0.25 250 0.25 ?
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2.
Lowry Assay ( Folin -Phenol Method ) (Folin Folin-Phenol
S HEPE HEPES Tris buffer 尿素Urea 胍Guanidine 硫酸钠 Sodium Sulfate 三氯乙酸 Trichloroacetic acid 乙醇Ethanol 丙酮Acetone
蛋白质的鉴定分析
Identification of proteins
10级硕士生高级生化实验技术课程表 20 2010
日期 授课内容 1．生化常用试剂的配制要求； 2．蛋白质分离纯化；介绍蛋白质的浓缩、结晶、保存。 （1）血浆IgG的分离纯化： 分段盐析、（透析）/SephadexG-50 凝胶过滤（脱盐） （2）亲和层析分离纯化工程菌中表达的GST融合蛋白 3．蛋白质的定量分析： 蛋白质的紫外定性、定量分析方法的选择及操作 lowry法/ BCA法/Bradford法等测不同溶液中蛋白质的含量 4．蛋白质的鉴定： （1）SDS-PAGE分离菌体蛋白及鉴定亲和层析纯化的GST融合蛋白 （2）细菌中GST融合蛋白表达的鉴定（Western Blotting ） 蛋白质分离纯化、性质鉴定分析(续)： Western Blotting( 续) 5. 酶学分析（酶活性测定及酶法分析） (1) 酶活性测定: 血浆/肝组织LDH酶活性测定 (2) LDH同工酶谱分析（琼脂糖凝胶电泳法） (3) 酶法分析： 酶法测定血清胆固醇、葡萄糖含量（终点测定法） Microplate reader在微量定量分析中的应用 实验总结、讨论 考试
Protein concentration (mg/ml)=1.45 A280 − 0.74 A260
② Warburg-Christian formula:
Protein concentration (mg/ml)=1.55 A280 − 0.76 A260
紫外分光光度法 UV spectrophotometry
凯氏微量定氮法 The Kjeldahl method
� The worldwide standard for calculating the protein content � 1 g nitrogen content is equal to 6.25 g protein. � Advantages: accurate � Disadvantages: complicated, low reproducibility Nitrogen in the sample is converted into ammonia by digestion process. The ammonia is distillated and collected. Then the amount of ammonia was quantified by titration and the initial protein concentration is then calculated.
Quantitative analysis of a pure sample ⑵ If the sample is a pure protein with known ε1mol/L or E1% at 280 nm, the amount of the protein can be quantified using the following formula:
� Advantages: High reproducibility � Disadvantages :
Interfering substance: Ammonium sulfate, Tris, etc. Low Sensitivity: 1～10 mg
双缩脲法 Biuret Test
Peptide Chains
酚类Phenols 柠檬酸Citric acid
Lowry Assay ( Folin -Phenol Method ) (Folin Folin-Phenol
Detectable range: 25~250 µg/ml
Simple Sensitive
Protein-toprotein variation More Interfering substances