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Flashcards in Protein Techniques Deck (30):
1

Insulin has ____side chains

2

2

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

110

3

Hemoglobin has ____side chains

4

4

Hexokinase has ____side chains

2

5

RNA polymerase has ____side chains

5

6

What do conjugated proteins contain

prosthetic groups

7

What does protein quantification rely on

Absorbance characteristics of aromatic amino acids

8

Free amino group where

At amino terminus

9

What is ion exchange chromotography

Separates on basis of electric charge

10

Anion exchange column has what?

Resin has fixed cationic groups and binds anionic substances

11

What does cation exchange column depend on

Resin has fixed anionic group and binds cations

12

What is affinity chromatography

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

13

What is molecular sieve chromatography

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

14

What is gel electrophoresis

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

15

What does SDS-PAGE stand for

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

16

What is Western blotting

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

17

What is isolelectric focusing

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.

18

What is 2D gel electrophoresis

1st dimension: Isoelectric focusing
2nd dimension: SDS-PAGE

19

What is Proteomics

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

20

What is Mass Spectrometry

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!

21

What are antibody-antigen interactions

Identifies specific proteins using antibodies

22

What are the techniques that use antibody antigen interactions

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.

23

What are antibodies

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

24

What are antigens

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

25

What is Protein sequencing

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

26

What if you have more than 50 aa?

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

27

What are Protease fragmentation examples:

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

28

Sequence proteins from ____terminus

amino; one at a time

29

Build proteins from ___terminus

C (carboxyl); opposite direction

30

What are the steps of protein synthesis

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