Protein Techniques

Question 1

Insulin has ____side chains

Answer 1

2

Question 2

To calculate MW you need to multiply the # of amino acids by ____

Answer 2

110

Question 3

Hemoglobin has ____side chains

Answer 3

4

Question 4

Hexokinase has ____side chains

Answer 4

2

Question 5

RNA polymerase has ____side chains

Answer 5

5

Question 6

What do conjugated proteins contain

Answer 6

prosthetic groups

Question 7

What does protein quantification rely on

Answer 7

Absorbance characteristics of aromatic amino acids

Question 8

Free amino group where

Answer 8

At amino terminus

Question 9

What is ion exchange chromotography

Answer 9

Separates on basis of electric charge

Question 10

Anion exchange column has what?

Answer 10

Resin has fixed cationic groups and binds anionic substances

Question 11

What does cation exchange column depend on

Answer 11

Resin has fixed anionic group and binds cations

Question 12

What is affinity chromatography

Answer 12

Based on binding specificity.

a. Resin is cross linked with a specific ligand
b. Protein to be purified will bind tightly
c. all other protein is washed away
d. Purified protein is released by addition of excess ligand

Question 13

What is molecular sieve chromatography

Answer 13

Based on SIZE

a. Resin is comprised of beads with tiny pores
b. Large proteins cant enter beads and fall through column quickly
c. Small proteins are trapped and move slowly, while BIG things come out quickly

Question 14

What is gel electrophoresis

Answer 14

Separates on basis of size

a. SDS imparts overwhelming negative charge on proteins
b. Proteins will migrate towards (+ pole)
c. Largest move slowly, smallest moves faster

Question 15

What does SDS-PAGE stand for

Answer 15

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 16

What is Western blotting

Answer 16

Transfer to a membrane (paper filter) –>designed to bind proteins. Pushes protein out of gel and onto paper. Soak blot in specific antibody solution.

Question 17

What is isolelectric focusing

Answer 17

Separates on basis of isolectric point
a. pH gradient is produced by allowing a mixture of ampholytes in PAGE to migrate to neutral positions in an electric field.

Question 18

What is 2D gel electrophoresis

Answer 18

1st dimension: Isoelectric focusing

2nd dimension: SDS-PAGE

Question 19

What is Proteomics

Answer 19

Uses 2D PAGE and Mass Spec to identify differences in protein profile of two cell populations

a. Shine light on cell; cells respond differently and start making different proteins based on light dosage
b. Label them with 2 diff dyes that will coalesce. Take two pops and mix them together and do 2D gel electrophoresis. When you expose them to infrared light you can see which ones are present in treatment group and not at all

Question 20

What is Mass Spectrometry

Answer 20

a. Proteins are added to a light absorbing matrix, then pulsed with laser which ionizes the proteins
b. Proteins are desorbed from matrix and enter into vacuum.
c. Charged proteins introduced into magnetic fields
d. Path through field is a function of mass to charge ration
- Patterns are very specific!

Question 21

What are antibody-antigen interactions

Answer 21

Identifies specific proteins using antibodies

Question 22

What are the techniques that use antibody antigen interactions

Answer 22

1. Immunoblots (Western Blots): Transfer of proteins from gel electrophoresis onto filter paper and soaking blot with antibody antigen solution
2. ELISA: Plate is precoded with antibodies. Can put protein in solution. If protein your interested in is in solution it will bind to antibody and cause color change.
3. Immunocytochemistry: Usually done with cells on culture. Using antibody to determine if a specific protein is present in tissue or cells that you’re working on.

Question 23

What are antibodies

Answer 23

Proteins produced by B lymphocytes (immuglobulins) to destroy antigen containing foreign invaders.

Question 24

What are antigens

Answer 24

Any kind of proteins that elecit an antibody response in your body.

Question 25

What is Protein sequencing

Answer 25

Based on Edman Degradation:

a. Anything less than 50 aa can be directly sequenced
b. Peptide bonds are hard to break (covalent bonds)
c. if you have phenylisothiocyanate it gives nice ring structure to detect aa spectrophotometrically
d. Cyanide group makes next bond very vulnerable
d. Can use mild hyrolyssis condition to cleave off aa.
e. Keep repeating

Question 26

What if you have more than 50 aa?

Answer 26

Must be fragmented prior to sequence analysis

1. Divide it into multiple samples
2. DO a.a. analysis to see how many sites there are for various digestive enzymes to cut it into 2/3 fragments.
3. React with FDNB; labels the N terminus only; separates aa’s
4. Reduce disulfide bonds and cleave with trypsin; separate fragements and sequences the fragments
5. Cleave with CNBr to separate fragments
6. Establish sequence

Question 27

What are Protease fragmentation examples:

Answer 27

1. Trypsin: cleaves on carbonyl side of basic amino acids (Lys or Arg)
2. Chymotrypsin: Carbonyl side of aromatics (Phe or Tyr)
3. S. aureus V8 protease: Carbonyl side of acidic a.a. (Glu or Asp)
4. Cyanogen Bromide: Carbonyl side of methionine

Question 28

Sequence proteins from ____terminus

Answer 28

amino; one at a time

Question 29

Build proteins from ___terminus

Answer 29

C (carboxyl); opposite direction

Question 30

What are the steps of protein synthesis

Answer 30

1. Bring in each aa with amino group protected so that it doesnt bind on amino side and binds with carboxy side.
2. Deprotect and bring in next aa that has been protected on amino terminus and activated on carboxy terminus
3. Repeat