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

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.

Rockefeller university

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.

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

The surfactant surrounds DNA fragments

sodium dodecylbenzene sulfonate

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

sodium dodecy sulfonate

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

Can surround an amino acid or “piece of DNA”

SDS Gel electrophoresis

The uv transilluninator

Dr. Alfrey—SDS gel electrophoresis of mouse leukemia cells treated with topo II isomerase poisons

It is similar to Paper chromatography! 

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

He would put me to sleep here and there!

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

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.

Cancer medications!

Bleomycin Adriamycin Tamoxiphen Estrogen Hormone

therapy!

He said “I study SDS Gel electrophoresis”

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

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

I said, “I’ll listen!”

These Columns have electric fields to migrate portions of DNA

SDS Gel electrophoresis: an Assay

SDS Gel electrophoresis: a series of steps!

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,

The bowling lane!

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.

The matrix: Poly Acrylimide (nail polish)

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

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.

isoleucine

aspartic acid

arginine

Alanine

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.

technique of electrophoresis

technique of electrophoresis

The isoelectric points

The gel is “charged”

The amino acids are“charged”

Can separate proteins

Can also manipulate DNA

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).

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.

And, detection with UV light

So, I can help those with cancer!

And, help kids play another day!

And, we can have some fun too!

Eating right!

Getting some excercise

No smoking! No drinking!

Or, meet the “five”