Lab 2 - Comparative Proteomics

Question 1

What is Protemics?

Answer 1

The study of proteins, particularly their structures and functions

Question 2

What is the proteome of an organism?

Answer 2

The entirety of proteins in existence in an organism throughout its life cycle.

Question 3

What is cell type?

Answer 3

The entirety of proteins found in a particular cell type under a particular type of stimulation.

Question 4

What did we use to examine proteins from closely and distantly related fish and to identity similarities and differences in the organisms’ protein profiles?

Answer 4

SDS-PAGE electrophoresis

We collected electrophoresis data.

Question 5

What is a cladogram?

Answer 5

A fish family tree

It can be constructed based on protein bands the fish have in common.

Question 6

What is cladistic analysis?

Answer 6

It assumes that when 2 organisms share a common characteristic, they also share a common ancestor with that same characteristic.

Question 7

What drives our (most familiar daily) movements?

Answer 7

The interactions between specialized proteins in our muscle fibers.

Question 8

What are the basic contractile elements of the muscle cell?

Answer 8

The myofibrils that are bundled into muscle fibres.

Question 9

What does each myofibril consist of?

Answer 9

A linear series of contractile units called sarcomeres.

Question 10

Sarcomeres are precisely arranged assemblies of:

Answer 10

actin and myosin protein filaments

(major components in muscle tissue)

Thin filaments of actin are aligned with thick filaments of myosin in parallel and partly overlapping manner. The combined contraction of many sarcomeres along a muscle fiber causes contraction of the entire muscle.

Question 11

Actin an myosin are highly conserved across all:

Answer 11

animal species

Question 12

Polyacrylamide gel electrophoresis (PAGE) can be used:

Answer 12

to separate small molecules such as proteins

Question 13

Prior to electrophoresis, the proteins are treated with what? And to do what?

Answer 13

The detergent sodium dodecyl sulfate (SDS) and heat. They denature the protein tertiary and quaternary structures to make the proteins more linear. SDS also gives the protein an overall negative charge.

Question 14

What does the power supply generate across the gel between the two electrodes?

Answer 14

A voltage gradient

Question 15

As soon as the electric current is applied,

Answer 15

the SDS-coated proteins begin their race toward the positive electrode. The smaller more quickly than the larger ones, so over time, the proteins will be separated according to their size.

Question 16

Protein size is measured in:

Answer 16

daltons (D), a measure of molecular mass

1 dalton = mass of a H atom

Question 17

What was the purpose of filling a Fish data sheet?

Answer 17

Form fits function, so depending on the fish’s biology, environment and swim type, the proteins are likely specific.

Question 18

What was the role of Tris-HCl in the protein extraction and eventual separation via PAGE of the fish samples?

Answer 18

The buffering solution is capable of extracting soluble cytoplasmic proteins.

Question 19

What was the role of SDS in the protein extraction and eventual separation via PAGE of the fish samples?

Answer 19

This denatures tert and quart structures of the proteins and confers a negative charge to them.

Question 20

What was the role of bromophenol blue in the protein extraction and eventual separation via PAGE of the fish samples?

Answer 20

This is a tracking dye that is highly mobile.

Question 21

What was the role of Diothiothreitol (DTT) in the protein extraction and eventual separation via PAGE of the fish samples?

Answer 21

DTT prevents intramolecular/intermolecular disulphide bonds from forming between cysteine residue of proteins.

Question 22

What was the purpose of agitating (flicking and vortexing) the samples in the extraction process?

Answer 22

To homogenize samples

Question 23

What was the purpose of heating (95 degree C) the samples in the extraction process?

Answer 23

This ensures denaturation of proteins and redissolves any precipitated detergent.

Question 24

Why do SDS-coated proteins move when placed in an electric field?

Answer 24

They move because they are negatively charged, away from the negatively charged cathode and towards the positively charged anode.

Question 25

What is the purpose of actin and myosin standards and the Precision Plus Protein Kaleidoscope prestained standard?

Answer 25

Actin and myosin are common to animals. The kaleidoscope has many proteins with known molecular mass. The 2 standards act as controls and are used to compare our unknown proteins.

Question 26

Which protein migrates farthest? Why?

Answer 26

The shorter/lighter proteins will migrate farthest because their smaller size allows them to more easily pass between the gel molecules, acting as a sieve.

Question 27

Would the migration of proteins on the gel been affected if SDS was not added to the samples?

Answer 27

SDS is used to stabilize negative charges on proteins. Without it, the movement of proteins wouldn’t necessarily be consistent. The same protein might move in different directions in different samples.

Question 28

Would Stain Free SDS-Page technique be able to identify every single protein in the sample? Would two proteins that travelled the same distance from the sample well be absolutely the same?

Answer 28

Not necessarily. It only means they have the same molecular weight. They could have different types of amino acids, which determines the protein’s nature.

Question 29

What accounts for the differences between our cladogram and the simplified cladogram (evolutionary tree)?

Answer 29

We made ours based on protein, while the tree provided by the University of Arizona is based off of DNA.