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Flashcards in Biotech Final - Lab 3 Deck (16)
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1

The electrophoresis of proteins using polyacrylamide gels allows for the separation of proteins into their ______ _______.

Individual subunits

2

What strong anionic detergent is used in SDS-PAGE?

SDS

3

What is used to reduce proteins to their primary structure?

SDS, heat and reducing agents.

4

How does SDS denature proteins?

By "wrapping around" the polypeptide backbone binding to the protein in a mass ratio of 1.4:1. SDS also disrupts hydrogen bonds, blocks hydrophobic interactions and substantially unfolds proteins.

5

What does DDT do?

DDT reduces disulphide bonds.

6

The SDS-denatured and reduced proteins are flexible rods with uniform _____ charge per _____ _____.

Negative charge per unit length

7

The larger the complex, the _____ the migration in the SDS sieving gel.

Slower

8

The separation ability of an SDS-PAGE gel is contigent on the concentration of ______ and the amount of ______-______ in a gel.

polyacrylamide and cross-linking.

9

What does bis-acrylamide do?

Provides rigidity and tensile strength to the gel and forms pores, allowing SDS:protein complexes to pass through them.

10

The size of the pores in an SDS-PAGE gel decrease as?

The ratio of bis-acrylamide: acrylamide increases.

11

What buffer system did we use?

Discontinuous.

12

What is a discontinuous buffer system?

Buffer in the reservoirs and the sample have a different pH and ionic strength than the buffer used to cast the gel.

13

What does a discontinuous buffer do?

Effectively concentrates the protein into a small volume allowing for increased resolution.

14

How is the SDS-PAGE gel stained?

Using either Coomassie Blue or Silver staining. Coomassie Brilliant Blue dye forms strong complexes and stoichiometrically binds with proteins, but does not bind tightly to the SDS-PAGE gel.

15

Proteins separated by SDS-PAGE are fixed using?

methanol or ethanol:glacial acetic acid, then stained using Coomassie Blue.

16

The uptake of dye is proportional to what?

The amount of protein.

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