BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu


Lecture 20

Gel Electrophoresis


Gel electrophoresis is used to characterize one of the most basic properties - molecular mass - of both polynucleotides and polypeptides

DNA:
The most common gel electrophoresis materials for DNA molecules is

DNA agarose gels

The electrophoretic migration rate of DNA through agarose gels is dependent upon four main parameters:

1. The molecular size of the DNA. Molecules of linear duplex DNA travel through agarose gels at a rate which is inversely proportional to the log of their molecular weight.

Mr 1/log (Mw)

Example: Compare molecular mass vs. expected migration rate:

Molecular Mass (Da)

log (Molec. Mass)

1/log (Molec. Mass)
i.e. relative Mr

100,000

5.0

0.20

50,000

4.7

0.21

10,000

4.0

0.25

5,000

3.7

0.27

1,000

3.0

0.33

2. The agarose concentration. There is an inverse linear relationship between the logarithm of the electrophoretic mobility and gel concentration.

log (Mr) 1/[gel]

Gel %

1/Gel %

inv log(1/Gel %)
(i.e. relative Mr)

2.0

0.50

3.2

1.5

0.67

4.6

1.0

1.00

10.0

0.5

2.00

100.0

3. The conformation of the DNA.

These different forms of the same DNA migrate at different rates through an agarose gel.

4. The applied voltage.

Agarose (%)

Range of separation of linear DNA

(in kilobases)

0.3

60 - 5

0.6

20 - 1

0.7

10 - 0.8

0.9

7 - 0.5

1.2

6 - 0.4

1.5

4 - 0.2

2.0

3 - 0.1

Finally, the DNA being an acidic molecule, migrates towards the positively charged electrode (cathode).

DNA acrylamide gels

Gel Electrophoresis for Proteins

Acrylamide (%)

Range of separation of Polypeptides

(in kilodaltons)

8

200 - 25

10

100 - 15

12.5

70 - 10

15

60 - 6

20

40 - 4

Stains for DNA

Ethidium

Because ethidium is a DNA intercalating agent, it is a powerful mutagen. Incorporation of ethidium in the DNA of living organisms (i.e. you and I) can cause (unwanted) mutations.

Note:

Flourescence shadowing:

Stains for Proteins

Coomassie brilliant blue

Silver stain

Molecular Weight Standards

Protein

Molecular Mass (Da)

Phosphorylase B

94,000

Bovine Serum Albumin

67,000

Ovalbumin

43,000

Carbonic Anhydrase

30,000

Soybean Trypsin Inhibitor

20,100

a-Lactalbumin

14,400


1998 Dr. Michael Blaber