Topic 6 - Biochemical Methods Flashcards Preview

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Flashcards in Topic 6 - Biochemical Methods Deck (25)
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1
Q

What is an analyte?

A

what you are analysing (jargon)

2
Q

Describe tissue homogenisation & cell lysis

A

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

3
Q

What happens in differential centrifugation?

A

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

4
Q

What is chromotography used for?

A

to isolate & purify enzyme from other proteins in nucleus

5
Q

Describe column chromotography. What are the two phases?

A

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

6
Q

What are the 3 kinds of column chromatography?

A

ion exchange

size-exclusion

affinity

7
Q

Describe ion exchange chromatography

A

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

8
Q

Describe size-exclusion chromatography

A

is a “molecular sieve”.

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

9
Q

Describe affinity chromatography

A

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
_

10
Q

What is the ELISA test?

A

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

11
Q

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

A

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.

12
Q

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

A

Tryptophan and Tyrosine

13
Q

Briefly describe electrophoresis and name the main type

A

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

14
Q

Describe SDS-PAGE method

A

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

15
Q

Describe the process of Western Blotting

A

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)
16
Q

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

A
17
Q

What is Mass Spectrometry and what is it used for?

A

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

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

18
Q

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

(view pic in answer)

A

useful in determining cell functions as recombinant proteins are tagged

any aa sequence at all can be created & tested for

19
Q

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

A
  • 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.
20
Q

Fluorescence can be used to determine…?

A

protein location in vivo

21
Q

A bit about Green Fluorescent Protein (GFP) ?

A

use recombinant DNA tech. to attach GFP to POI

visualise w/ a fluorescent microscope

22
Q

A bit about Immunofluorescence

A

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

23
Q

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?

A

true

24
Q

Advantages and disadvantages of using fluorescence to ascertain protein function

A

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
25
Q
A