Topic 6 - Biochemical Methods

Question 1

What is an analyte?

Answer 1

what you are analysing (jargon)

Question 2

Describe tissue homogenisation & cell lysis

Answer 2

a tissue is homogenised in ice cold 0.25M sucrose solution -treatment ruptures cell membranes, leaves organelles intact Can then be separated into subcellular fractions Adding a detergent will lyse cell membranes (organelles not intact -allows access to protein =>creates homogenate

Question 3

What happens in differential centrifugation?

Answer 3

Organelles differ in size and density, therefore sediment at different rates

Question 4

What is chromotography used for?

Answer 4

to isolate & purify enzyme from other proteins in nucleus

Question 5

Describe column chromotography. What are the two phases?

Answer 5

Stationary phase

Mobile phase: a buffered solution is allowed to move through stationary phase

• buffer can be moved under gravity or pumped at high pressure
• buffer is constantly replaced

Efffluent is collected in fractions or constantly monitored

Individual proteins move at different rates depending on properties & interactions w/ stationary phase

Question 6

What are the 3 kinds of column chromatography?

Answer 6

ion exchange

size-exclusion

affinity

Question 7

Describe ion exchange chromatography

Answer 7

relies on differences in electrostatic attraction of molecules to a charged stationary phase; separation on basis of charge

Question 8

Describe size-exclusion chromatography

Answer 8

is a “molecular sieve”.

separation based on capacity if molecules to penetrate inside porous stationary phase

Question 9

Describe affinity chromatography

Answer 9

relies on specific binding b/w a small molecule and a macromolecule (usually protein)

_separates proteins according to their ability to bind strongly to specific molecules (ligands)
on surface of beads_

Question 10

What is the ELISA test?

Answer 10

Enzyme-Linked Immunosorbent Assay

-Detection of protein involves antigen-antibody recognition, using an antibody with an attached enzyme.

In this case, antigen (protein) is in solution and binds to immobilised antibody (e.g. bound to plastic wells)

-attached enzyme produces colour if substrate is added

Question 11

Specific activity can be used to assess enzyme purity. Discuss.

Answer 11

1.0 unit of enzyme activity is defined as the amount of enxyme causing transformation of 1 mmol of substrate to product per minute at 25˚C.

Specific activity is defined as the number of enzyme units per milligram (mg) of total protein

During purification, V decreases at each step but specific act. increases as you remove unwanted proteins/biomolecules at each step.

Question 12

aromatic aa’s absorb light in the UV region. What are the two strongest chromophores?

Answer 12

Tryptophan and Tyrosine

Question 13

Briefly describe electrophoresis and name the main type

Answer 13

a porous gel matrix has lots of channels running through it.
-larger protein moves more slowly
Proteins can travel along these channelsin direction of electric field separated by size and/or charge.

Main type SDS-PAGE

Question 14

Describe SDS-PAGE method

Answer 14

SDS: sodium dodecyl sulfate -saturates proteins and seperates according to size

PAGE: Polyacrylamide gel electrophoresis -separates proteins & nucleic acids on basis of size & charge

SDS binds to & unfolds proteins, gives them -ve charge

Question 15

Describe the process of Western Blotting

Answer 15

Used to ID and/or quantify purified proteins from a mixture

1. seperate proteins using SDS PAGE
2. transfer proteins to membrane (imprint)
3. incubate membrane w/ an antibody directed against protein of interest
4. Use 2nd antibody tp bind 1st antibody. This antibody is linked to enzyme that can be detected by colour reaction (ELISA)

Question 16

Isoeletric focusing can be used to separate proteins by pI. how?

Answer 16

Question 17

What is Mass Spectrometry and what is it used for?

Answer 17

MS breaks peptides down into mixtures of fragments or different sizes.

Is used to precisely ID mass of a peptide, and therefore aa sequence.

Question 18

DNA cloning => recombinant proteins. What is their use?

(view pic in answer)

Answer 18

useful in determining cell functions as recombinant proteins are tagged

any aa sequence at all can be created & tested for

Question 19

Polymerase Chain Reaction (PCR) is used for…? what are the general steps?

Answer 19

• used to amplify DNA (regions of interest & circular plasmids)
• clone DNA
• detect genes
• to mutate genes

1. Mix together target DNA, primers, nucleotides (dATP, dCTP etc), thermostable DNA polymerase
2. Place mixture into thermocycle
   - melt DNA at 95˚C (Heat to separate strands)
   - cool separated strands to about 50-60˚C
   - primers anneal target
   - polymerase extends primers in 5’-3’ direction
3. After a round of elongation is done, repeat.

Question 20

Fluorescence can be used to determine…?

Answer 20

protein location in vivo

Question 21

A bit about Green Fluorescent Protein (GFP) ?

Answer 21

use recombinant DNA tech. to attach GFP to POI

visualise w/ a fluorescent microscope

Question 22

A bit about Immunofluorescence

Answer 22

Tag protein w/ primary antibody & detect w/ secondary antibody containing fluorescent tag

Question 23

When exposed to appropriate light, a fluorophore will absorb light at a characteristic wavelength and then emit light at a longer (lower energy) wavelength which is detectable.
T or F?

Answer 23

true

Question 24

Advantages and disadvantages of using fluorescence to ascertain protein function

Answer 24

Adv:

• not hazardous
• different fluorophores can be imaged in same cell

Disadv:

• can be sensitive to pH & temp
• can be excited by light and cause photobleaching