Chapter 5 Molecular Tools for Studying Genes and Gene Activity

1. Chapter 5 Molecular Tools for Studying Genes and Gene Activity Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu

2. Electrophoresis • Agarose gel electrophoresis

3. Separates DNA (or RNA or Protein) fragments on the basis of charge and size • Because DNA is an acid, it looses protons in basic buffers; thus it has a negative charge that is uniform per unit length • Agarose (a polysaccharide) or other gel matrices are difficult for large DNA fragments to move through • The larger the fragment, the more difficulty it has moving through gels • By placing DNA in a gel, then applying a voltage across the gel, the negatively charged DNA will move toward the anode (positive electrode) • Large fragments lag behind while small fragments move through the gel relatively rapidly

4. Gel Electrophoresis - Wells Direction of DNA Travel + Large Small

5. Text Art Page 91 The electrophoretic mobility of a DNA fragment is inversely proportional to the log of its size.

6. Figure 5.2b 3 mm 10 20 30 40 50 60 70

7. Figure 5.1b

8. Agarose gel electrophoresis

9. Electrophoresis • Agarose gel electrophoresis • PFGE

10. + + + + + | | | | | But, what if you want to separate larger fragments, such as entire yeast chromosomes? Pulsed-field gel electrophoresis (PFGE) can resolve fragments from 200 Kpb (0.2 Mbp) to 6000 Kbp (6 Mbp).

11. Figure 5.3 2.2 Mbp 0.2 Mbp

12. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE

13. Electrophoresis of proteins using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) • Denaturing electrophoresis • Detergent (SDS) • Reducing agent (b-mercaptoethanol) • Heat • SDS binds to denatured proteins, making them negatively charged • Migrate through gel based on size • Molecular weight markers allow for estimation of size of polypeptide • Modifications (e.g., glycosylation) can significantly impact the apparent size of the protein

14. Polyacrylamide gels are composed of chains of polymerized acrylamide that are cross-linked by a bifunctional agent • N,N’-methylene-bis-acrylamide • Size of pores decrease as the ratio of bisacrylamide:acrylamide increases, reaching a minimum at ~1:20 ratio • A 1:29 ratio is most commonly used, as it is capable of resolving polypeptides that differ is size by as little as 3%

15. Electrophoresis of proteins using SDS-PAGE Molar ratio of bisacrylamide:acrylamide is 1:29

16. Figure 5.4

17. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels

18. + + Separated by isoelectric point Acidic Separated by Size Proteins stop migrating when they reach their isoelectric point (pH at which they have no net charge) Isoelectric focusing Basic | | Standard SDS polyacrylamide gel

20. Treat cells with a drug Treat cells with vehicle (control) Label proteins with Cy5 (blue) Label proteins with Cy3 (red) 2D-gel 2D-gel Overlay gels Analyze for differences

21. Figure 5.5

22. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography

23. Low Salt High Salt DEAE-Sephadex (Positively Charged) Negatively charged proteins bind to resin (the stronger the charge, the tighter the binding) Tighter bound protein elutes with higher concentrations of salt Weakest bound protein (weakest negative charge) comes off first.

24. Figure 5.6

25. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography • Gel filtration chromatography

26. Figure 5.7a

27. Buffer Sephadex Smaller proteins elute with additional buffer Largest proteins come off first (void volume).

28. Figure 5.7b

29. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography • Gel filtration chromatography • Autoradiography • X-ray film

30. Figure 5.8

31. X-ray Film Cassette Intensifying Screen: •b-emitters only •(3H, 14C, 35S, 32P) •-70°C or cooler X-ray Film Nitrocellulose or Nylon Membrane

32. Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag 32P 32P Intensifying screen (fluoresces with b-rays)

33. Figure 5.9 Densitometry

34. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography • Gel filtration chromatography • Autoradiography • X-ray film • Phosphorimaging

35. X-ray film cassette Storage Phosphor plate Nitrocellulose or Nylon Membrane

36. Phosphorimaging is much more sensitive than X-ray film • <0.95 dpm/mm2 for 1 hr exposure to 14C • <0.15 dpm/mm2 for 1 hr exposure to 32P • Dynamic range of 5 orders of magnitude • Shorter exposure times (50-90%)

37. Figure 5.10

38. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography • Gel filtration chromatography • Autoradiography • X-ray film • Phosphorimaging • Liquid scintillation counting

40. Electrophoresis • Agarose gel electrophoresis • PFGE • SDS-PAGE • 2-D gels • Other types of chromatography • Ion-exchange chromatography • Gel filtration chromatography • Autoradiography • X-ray film • Phosphorimaging • Liquid scintillation counting • Non-radioactive tracers

41. Figure 5.11

42. Electrophoresis • Other types of chromatography • Autoradiography • Nucleic acid blotting • Southern blotting

43. Figure 5.12

44. Southern blot: transfer of DNA from a gel to a solid support membrane Northern blot: transfer of RNA from a gel to a solid support membrane

45. Electrophoresis • Other types of chromatography • Autoradiography • Nucleic acid blotting • Southern blotting • DNA fingerprinting

46. Figure 5.13

47. Figure 5.14

48. Figure 5.15

49. Electrophoresis • Other types of chromatography • Autoradiography • Nucleic acid blotting • Southern blotting • DNA fingerprinting • Northern blotting

50. Figure 5.16