Question

What is Coomassie blue? How does it function?

(1.5 marks)

As an alternative to coomassie blue staining, a silver nitrate solution can be used. How does silver nitrate

staining differ and under which situations might it be used?

In which ways are vertical polyacrylamide gels and horizontal agarose gels the same? In which ways do

they differ?

5

In which loaded fraction (lane) would you expect to find the most proteins? The least? Explain

6

In your own words, provide a brief overview of next week’s lab. Include the experi

Answer 1

Coomassie Brilliant blue (CBB) is a stain that is used in PAGE gels. The sulfonic acid group of the stain ofrms ionic bonds with the amino group of proteins. Apart from this, there is also weak vander waals interaction between the dye and proteins. The sensitivity of the detection of proteins in polyacrylamide gels by CBB depends upon the type of dye used: CBB R-250 can detect about 100 ng protein whereas CBB G-250 is much more sensitive as it would detect ~30 ng protein.

Silver nitrate staining of proteins in PAGE gels works on the principle where soluble silver is reduced to metalic silver when protein molecules interact with it. This method is much more sensitive as it detects as low as 1 ng protein. This method can be used to detect proteins that are present in trace amounts in a sample.

Vertical PAGE gels and horizontal agarose gels both separate DNA on the basis of their size, as both form a porous matrix for the separation of macromolecules. Both Agarose and PAGE gel concenrations can be varied to better separate (increase or decrease the distance between bands)  molecules based on their size. In both agarose and PAGE gels, buffer systems can be varied to manipulate resolution of molecules and duration of electrophoresis.

PAGE gels and Agarose gels differ in that Agarose is a naturally occuring polysaccharide derived from sea weeds whereas poly acrylamide is a mixture of acrylamide and bis-acrylamide that are chemically cross-linked and polymerised using Ammonium persulfate and TEMED or by photopolymerization with the aid of riboflavin. Compared to agarose gels, PAGE gels give better resolution of molecules. Agarose gels are harmless unless ethidium bromide is added to the gel. Acryamide is a neurotoxin and so careful handling of the gels is necessary; the use of ethidium bromide in PAGE gels require additional care in handling the gels. Agarose gels take much less time to prepare and they solidify easily. PAGE gels require longer wait times for the gels to set. Also, PAGE gels tend to form bubbles while casting them so care should be taken while pouring the polymer mixture inbetween glass plates; agarose requires no such care. DNA as small as 5 bp can be resolved in a PAGE gel whereas agarose gels cannot be used for DNA fragements less than 200 bp.