生物化学教学课件：Protein characterization

1、1Protein characterization2017-10-122Gel electrophoresis 凝胶电泳3SDS(Sodium Dodecyl Sulfate 十二烷基磺酸钠) Sodium dodecyl sulfate, CH3(CH2)10CH2-SO4-, Na+ b-mercaptoethanolHS-CH2CH2-OHDTT (dithiothreitol)Sth to know by the way:4SDS(PolyAcrylamide Gel Electrophoresis聚丙烯酰胺凝胶电泳) % GelMass Range750 kDa - 500 kDa1

2、020 kDa - 300 kDa1210 kDa - 200 kDa153 kDa - 100 kDa/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htmpolymerizeAcrylamide is neurotoxic!5Visualization of protein on gelCoomassie brilliant blue stainingSilver staining 银染考马斯亮蓝染色Involving the reduction of Ag+ to Ag;10-50 times sensitive tha

3、n coomassie blue.6IEF (isoelectric focusing gel等电聚焦电泳)7Two-dimensional gel (2D-gel)8Western blot （免疫印迹）SDSWestern blot9Mass spectrometry 质谱m/z molecular weight; error 100 ppm. ()10/wikipedia/commons/1/1f/Mass_spectrometry_protocol.pngPowerful modern mass spectrometry11Methods to quantify proteinUV a

4、bsorbance: A = e c l(Lambert-Beer law)absorbancecoefficientconcentrationlength of light pathGenerally, OD280 = 1 for 0.5 2 mg/ml proteinStaining methodsBiuret method, Lowry method, Bradford method, etc. 12Edman degradation sequencing13PTH Amino Acid StandardResidue LysEdman degradation sequencing14N

5、CEdman sequencing = N-terminal sequencing Need to cut protein into limited number of fragments. 15Protease digestion to fragment proteinstrypsinchymotrypsinpepsinGlu-C, Lys-C, Arg-C, Asp-N, etc;Cyanogen bromide (CNBr)C-termini of Tyr, Phe, and Trp. Leu, Met, Ala, Asp, and Glu are cleaved at a lower

6、rate. C-termini of Lys and Arg.Chemical cleavage at the C-terminus of Met.Mainly at C-termini of Phe and Leu. 16NC17Overlapping to get complete sequenceTTCC18Three-dimensional structure determinationX-ray crystallographyNMR (Nuclear Magnetic Resonance )EM (Electronic microscopy)Structure in solution;For small proteins ( 20 KD);For large proteins or protein assemblies;Relatively low resolution.19To know protein purity and sizeTo know protein structuresElectrophoresis (SDS, IEF, 2D gel, etc.)Mass spectrometryEdman degradation sequencingX-ray crystallography, etc.To q