Early Research of Topo II isomerase poisons carried out by Dr. Alfrey at Rockefeller University

1. The Rockefeller University
 SDS Agarose Gel Electrophoresis of Topo II
 Isomerase Poisons
 Student: Dr. Robert D. Craig, Ph.D
 Mentor: Dr. Vincent Alfrey, M.D, Ph.D

2.  The Rockefeller University is a private
university which focuses primarily on basic
research in the biomedical fields and offers
postgraduate and postdoctoral education. It
is located between 63rd and 68th Streets
along York Avenue, on the Upper East Side of
Manhattan in New York City, New York. Its
current president is Sir Paul Nurse.

3. Rockefeller university

4. Who works here???
 Twenty-three Nobel Prize winners have been
associated with the university.
 The university has been the site of many
important scientific breakthroughs.
Rockefeller scientists, for example,
established that DNA is the chemical basis of
heredity, discovered blood groups, showed
that viruses can cause cancer, founded the
modern field of cell biology, worked out the
structure of antibodies.

5. What happens when you Combine
soap with proteins?
 Both are zwitterions!
 Will form a clump! Or a miscelle
 Can use an electric field to guide down a
silicon column or gel

6. The surfactant surrounds DNA
fragments

7. sodium dodecylbenzene sulfonate
Sodium dodecylbenzene sulfonate -
Identification, toxicity, use,
water pollution potential,
ecological toxicity and
regulatory information

8. sodium dodecy sulfonate

9. Sodium Dodecyl Sulfate
 (SDS) SDS is the most common dissociating
agent used to denature native proteins to
individual polypeptides. When a protein
mixture is heated to 100 °C in presence of
SDS, the detergent wraps around the
polypeptide backbone. It binds to
polypeptides in a constant weight ratio of 1.4
g/g of polypeptide

10. Can surround an amino acid or “piece of
DNA”

11. SDS Gel electrophoresis

12. The uv transilluninator

13. Dr. Alfrey—SDS gel electrophoresis of mouse
leukemia cells treated with topo II isomerase
poisons
 It is similar to Paper chromatography!

14. This man, Dr. Alfrey, was really nice
and really famous!
 National Academy of Science

15. He would put me to sleep here
and there!

16. But, he took time out tell me some of
what he knew

17. This is what the famous guy told me!
 Topo II isomerase poisons are what your Mom
took!
 They are all we have right now!
 They stop all fast growing cells from
replicating
 That is why your Mom’s hair fell out
 That is why your stomach gets upset
 These medications stop fast reproducing
tissue.

18. Cancer medications!
 Bleomycin
 Adriamycin
 Tamoxiphen Estrogen Hormone therapy!

19. He said “I study SDS Gel
electrophoresis”

20. You can go “in the Lab”
Check out what I do!

21. He said, “listen to everything I say”, if
you want to know about cancer!

22. I said, “I’ll listen!”

23. These Columns have electric fields to
migrate portions of DNA

24. SDS Gel electrophoresis: an Assay

25. SDS Gel electrophoresis: a series of
steps!

26. technique of
electrophoresis
 The distribution of charged species in a sample
can be shown experimentally by observing the
movement of solute molecules in an electric
field, using the technique of electrophoresis. For
such experiments an ionic buffer solution is
incorporated in a solid matrix layer, composed of
paper or a crosslinked gelatin-like substance. A
small amount of the amino acid, peptide or
protein sample is placed near the center of the
matrix strip and an electric potential is applied at
the ends of the strip,

27. The bowling lane!

28. technique of electrophoresis
 The solid structure of the matrix retards the
diffusion of the solute molecules, which will
remain where they are inserted, unless acted
upon by the electrostatic potential. In the
example shown here, four different amino
acids are examined simultaneously in a pH
6.00 buffered medium.

29. The matrix: Poly Acrylimide
(nail polish)

30. A hard gel! You can control the movement of
DNA, and amino acids

31. technique of electrophoresis
 At pH 6.00 alanine and isoleucine exist on
average as neutral zwitterionic molecules,
and are not influenced by the electric field.
Arginine is a basic amino acid. Both base
functions exist as "onium" conjugate acids in
the pH 6.00 matrix.

32. isoleucine

33. aspartic acid

34. arginine

35. Alanine

36. technique of electrophoresis
 The solute molecules of arginine therefore
carry an excess positive charge, and they
move toward the cathode. The two carboxyl
functions in aspartic acid are both ionized at
pH 6.00, and the negatively charged solute
molecules move toward the anode in the
electric field.

37. technique of electrophoresis

38. technique of
electrophoresis

39. The isoelectric points

40. The gel is “charged”

41. The amino acids are“charged”

42. Can separate proteins

43. Can also manipulate DNA

44. Intercalating Agent
 After we have removed our gel from the running
tank, we need to use ultraviolet light to visualize
the DNA and verify that our Restriction Digest
was successful. Does DNA fluoresce when
exposed to UV light? Not on its own. Remember
when we added dyes to our Blue-Juice to help us
visualize the progress of the gel as it was
running? We do something similar in this
situation. Rather than "seeing" the DNA, we will
be "seeing" a different molecule, called Ethidium
Bromide (EtBr).

45. Intercalating Agent
 EtBr is an Intercalating Agent, meaning it
wedges itself into the grooves of DNA and stays
there. More base pairs mean more gooves, which
in turn means more EtBr can insert itself. This is
an important concept. EtBr also has the property
of fluorescing under UV light. So if we soak our
gel in a solution of EtBr, it will intercalate into
the DNA, then if we place our gel on or under a
UV source, we can "see" the DNA by actually
detecting the fluorescence of the EtBr. Wherever
there is DNA, we will see a bright band at that
point in the gel.

46. And, detection with UV light

47. So, I can help those with cancer!

48. And, help kids play another day!

49. And, we can have some fun too!

50. Eating right!

51. Getting some excercise

52. No smoking! No drinking!

53. Or, meet the “five”