Methods of Gel Electrophoresis Flashcards

1
Q

How is agarose gel prepared?

A

Add agarose to the required percentage in TAE buffer. Dissolve agarose by boiling and pour into a casting tray to let it cool down and solidify.

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2
Q

What is the composition of the loading buffer used in SDS-Polyacrylamide Gel Electrophoresis of Proteins?

A

Loading buffer contains glycerol and tracking dye: bromophenol blue. Also contains SDS and DTT or β-mercaptoethanol.

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3
Q

How are samples treated before loading in Non-reducing Polyacrylamide Gel Electrophoresis of Proteins?

A

Samples boiled for a few minutes in loading buffer.

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4
Q

Which electrode do molecules move towards in Native Polyacrylamide Gel Electrophoresis of Proteins?

A

Positive.

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5
Q

What is the principle of biomolecule separation in Agarose Gel Electrophoresis of DNA?

A

Agarose polymer forms a meshwork allowing DNA separation by size.

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6
Q

Which stain is commonly used to observe bands in SDS-Polyacrylamide Gel Electrophoresis of Proteins?

A

Coomassie blue.

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7
Q

Describe the process of gel preparation in SDS-Polyacrylamide Gel Electrophoresis of Proteins.

A

There are usually two layers, the stacking gel and the resolving gel. Both gels contain Acrylamide and bis-acrylamide. To cause “gelling,” one uses TEMED to catalyze the formation of persulfate radicals from ammonium persulfate. The radicals will cause acrylamide and bisacrylamide to cross-link leading to solidification of the gel.

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8
Q

What is the treatment of samples prior to loading in Native Polyacrylamide Gel Electrophoresis of Proteins?

A

Samples boiled for a few minutes in loading buffer.

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9
Q

How are proteins separated in Non-reducing Polyacrylamide Gel Electrophoresis of Proteins?

A

Protein is denatured and coated with negatively charged SDS which masks their intrinsic charges. Mobility is a linear function of the logarithms of their molecular mass.

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10
Q

Which tracking dye is commonly found in loading buffer used in Agarose Gel Electrophoresis of DNA?

A

Bromophenol blue.

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