《分子生物学 Molecular Biology》课程教学课件（PPT讲稿，英文版）Chapter 05 Molecular Tools for Studying Genes and Gene Activity

Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display. Chapter 5 Molecular Tools for Studying Genes and Gene Activity Peter Dazeley/Stone/Getty

Chapter 5 Molecular Tools for Studying Genes and Gene Activity

5.1 Molecular Separations Gel electrophoresis(凝胶电泳) staining with a fluorescent dye and the gel is examined under ultraviolet DNA is negatively charged cathode illumination The gel with slots DNA migrates toward anode agarose Figure 5.1 DNA gel electrophoresis. anode向 Mobility:Friction force In neutral pH buffer

Mobility: Friction force In neutral pH buffer 5.1 Molecular Separations Gel electrophoresis(凝胶电泳) The gel with slots DNA is negatively charged anode cathode staining with a fluorescent dye and the gel is examined under ultraviolet illumination agarose _ + anode

The mobilities of these fragments are plotted versus the log of their molecular weights (or number of base pairs) 2000p 1500 deviates strongly from linearity if the DNA is very large (mm Any unknown DNA can be electrophoresed in parallel with the standard fragments,and its size can be estimated if it falls within the range of the standards

logarithmic The mobilities of these fragments are plotted versus the log of their molecular weights (or number of base pairs) Any unknown DNA can be electrophoresed in parallel with the standard fragments , and its size can be estimated if it falls within the range of the standards deviates strongly from linearity if the DNA is very large

For the DNAs in the size ranges ~Mb(million base pairs,megabases) pulsed-field gel electrophoresis (脉冲凝胶电泳，PFGE) Instead of a constant current through the gel this method uses pulses of current with relatively long pulses in the forward direction and shorter pulses in the opposite,or Figure 5.3 Pulsed-field gel electrophoresis of yeast chromosomes. even sideways, direction·

For the DNAs in the size ranges ~Mb(million base pairs, megabases) pulsed-field gel electrophoresis (脉冲凝胶电泳,PFGE) Instead of a constant current through the gel， this method uses pulses of current， with relatively long pulses in the forward direction and shorter pulses in the opposite，or even sideways， direction．

polyacrylamide gel electrophoresis (PAGE,聚丙烯酰胺凝胶电泳) Sodium dodecy1 sulfate(SDS,十二烷基磺酸钠) M,(kD) Denaturing the subunits so they no longer 250 bind to one another. 160 105 The SDS has two added advantages: It coats all the polypeptides with negative charges so they all electrophorese toward 35 the anode(正极). It masks the natural charges of the subunits,so they electrophorese according to 15 0 their molecular masses and not by their native charges. Figure 5.4 SDS-polyacrylamide gel electrophoresis Pre-stained protein markers

Pre-stained protein markers polyacrylamide gel electrophoresis （PAGE,聚丙烯酰胺凝胶电泳) Sodium dodecyl sulfate(SDS,十二烷基磺酸钠) Denaturing the subunits so they no longer bind to one another. The SDS has two added advantages: ✓It coats all the polypeptides with negative charges，so they all electrophorese toward the anode(正极). ✓ It masks the natural charges of the subunits, so they electrophorese according to their molecular masses and not by their native charges

Two-dimensional Gel Electrophoresis First Decreasing High resolution protein separations dimension Isoelectric focusing First step:Isoelectric focusing electrophoresis (FE,等电聚焦电泳)，separating protein based on charge or isoelectric point. gel is placed on SDS polyacrylamide gel. Second step:SDS-PAGE,separating based on size. Seeond dimension SDS polyacrylamide gel electrophoresis Decreasing

Two-dimensional Gel Electrophoresis High resolution protein separations Second step: SDS-PAGE, separating based on size. First step: Isoelectric focusing electrophoresis (IFE,等电聚焦电泳), separating protein based on charge or isoelectric point

Protein Two-dimensional Gel Electrophoresis Sample IEF SDS-PAGE Isoelectric focusing 8 8 +

- + Isoelectric focusing Two-dimensional Gel Electrophoresis

Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display -Cy3 Mixed 5 (Source:Courtesy of Amersham Pharmacia Biotech.)

SUMMARY High-resolution separation of polypeptides can be achieved by two-dimensional gel electrophoresis,which uses isoelectric focusing in the first dimension and SDS-PAGE in the second

SUMMARY High-resolution separation of polypeptides can be achieved by two-dimensional gel electrophoresis, which uses isoelectric focusing in the first dimension and SDS-PAGE in the second

Ion-exchange chromatography(离子交换层析)uses a resin (to separate substances according to their charges. Fractionation of proteins by charge Elution buffer lon-exchang默resin protein mixture Removed the substances that had bound to the resin in (+ the column by passing a solution of gradually increasing beads with K ionic strength positive charge through the column. positively charged protein flows ④⊕④ through column ⊕⊕

Ion-exchange chromatography (离子交换层析) uses a resin （树脂）to separate substances according to their charges. Ion-exchange resin Elution buffer Removed the substances that had bound to the resin in the column by passing a solution of gradually increasing ionic strength through the column