Proteins: Determination of Primary Structure and Expression Flashcards Preview

Elements of Medicine > Proteins: Determination of Primary Structure and Expression > Flashcards

Flashcards in Proteins: Determination of Primary Structure and Expression Deck (28):
1

What are seven types of proteins with different functions?

Enzymes, hormones, transport, storage, structural, protective, contractile

2

What is the difference between a simple protein and a conjugated protein?

Simple protein is compose solely of amino acids while a conjugated protein has non AA components.

3

What are examples of conjugated proteins?

lipoproteins, glycoproteins, phosphoproteins, hemoproteins, flavoproteins, metalloproteins

4

How many amino acids are required for a protein to fold?

40

5

What is the common number of AA in proteins?

100-1000

6

How many genes in humans? How many expressed?

40,000 and 10k-15k

7

Some proteins are expressed at high levels and others at low. Give an example of each.

High: collagen
Low: hormones

8

Why are hormones harder to research than collagen?

Low expression makes identification difficult. Rely on animal hormones for research

9

What characteristics can be used to separate proteins?

solubility, charge, size, and affinity

10

Technique; Match to Characteristic
Ion Exchange Chromatography
Isoelectric focusing
Electrophoresis

Charge

11

Technique; Match to Characteristic
Dialysis and ultracentrifugation
Gel electrophoresis
Gel filtration

Size

12

Technique; Match to Characteristic
2D gel electrophoresis

Charge and size

13

Technique; Match to Characteristic
Affinity Chromatography

Specificity

14

Technique; Match to Characteristic
Paper Chromatography
Reverse-Phase Chromatography
Hydrophobic Chromatography

Polarity

15

In affinity chromatography, the _____ attaches to the stationary phase which is the ligand. This occurs in a lot of test tubes.

target

16

Why does SDS Page use sulfur?

Mask the charge of the molecules so that they all become negative. Allows samples to move toward positive charge based on MW alone. Speed at which they move toward positive indicates weight.

17

Western Blot (immunoblotting) occurs after what size filtration technique?

SDS Page or any size technique

18

In Western Blot, the gel samples are incubated with ______ _____ which are formulated to attach to the specific protein of interest. Then, labeled ______ _____ are added to attach to the _____ ______.

primary antibodies
secondary antibodies
primary antibodies

19

After the luminescent antibody binds, a colorimetry test can be used to detect the protein of interest. What is a colorimetry test?

A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light.

20

Does ELISA measure protein expression or activity?

Expression

21

What are the steps to completing an ELISA?

1. Coat surface with antigen.
2. Block unoccupied sites with DUMB protein
3. Add antibody for antigen
4. Add antibody for first antibody. This second antibody has an enzyme on it
5. Add substrate for enzyme
6. Test degree of color to see degree of enzyme expression

22

The primary structure of insulin was first determined by ______.

Sanger

23

What are the steps of insulin maturation?

1. Preproinsulin (A, B, C chains)
2. Proinsulin (cleavage of signal peptide of N term and disulfide bond formation)
3. Insulin (cleavage of C)

24

Edman Degradation uses ______ cleaving agents to create _______ amino acid sequence.

multiple, overlapping

25

N terminal amino acids are removed one at a time by binding to _________ or a derivative _____. This is a form of sequencing via ____ _____.

phenylisothiocyanate (Edman's reagent)
PTH
Edman Degradation

26

What are two forms of mass spec that can be used to identify proteins?

MALDI and ESI

27

Mutations in what form of insulin cause neonatal diabetes?

Preproinsulin

28

What are the benefits of using mass spec for protein identification?

High specificity even if protein isn't completely pure
Sensitivity allows for quantitative analysis
Can identify full peptides
Samples can be run in parallel for multiple proteins
Can determine post translational modifications with locations