Microbiology 4: (Patel) Protein analysis

Question 1

2 types of cultured cells? Advantages? Disadvantages?

Answer 1

Primary cultures
-> Cells derived directly from tissues e.g. blood,liver
+’s best experimental model of in-vivo cells
-‘s cells cease to grow after a few cell divisions

Cell lines
-> Treatment of primary cells with radiation (essentially cancer cells)
+’s cells continue to grow and divide indefinitely if proper conditions are maintained
-‘s Different from ‘normal’ cells

Question 2

5 methods of making cell lysate / lysing mammalian cells

Answer 2

Hypotonic Lysis
Homogenisation (disruption via grinding,shear forces)
Sonication (ultrasound)
Freeze-Thawing
Biological Detergents

Question 3

2 types of biological detergent used in lysis buffers and an example of each one?

Answer 3

Ionic Detergent e.g. SDS
- Strong, denatures proteins by binding to their internal ‘hydrophobic cores’, disrupts ionic interactions -> INACTIVATES PROTEIN

Non-ionic Detergent e.g. Triton-X100

• Mild, binds only to the membrane spanning domain of the proteins -> proteins REMAIN ACTIVE
• > solubilise proteins

Question 4

Typical composition of mammalian lysis buffer? Reason for each?

Answer 4

0.01M Tris-HCl pH 7.4 - pH buffer
0.15M NaCl - maintain ionic strength mimic phys. conditions
1% v/v detergent - lyse cells, solubilise proteins
Protease Inhibitors - prevent protein degredation

(MgCl2) - some enzymes dependent of Mg cation
(Phosphotase inhibitors) - prevent inactivation/activation of proteins via phosphorylation

Question 5

Chromatographic methods of protein extraction/separation?

Answer 5

Ionic exchange - separation according to charge

Hydrophobic chromatography - proteins separated according to hydrophobicity

Gel-filtration - separation according to size/molecular weight

Affinity chromatography - separation based on ability to bind to specific ligand immobilised to an insoluble support matrix)
- e.g., enzyme-substrate, antibody-antigen, hormone-receptor

Question 6

What is SDS-page gel electrophoresis and why is it useD?

Answer 6

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

• widely used because it can rapidly separate all types of proteins
• separates by size only

uses:

• protein identification (western blotting)
• determining sample purity
• identifying di-sulphide bonds
• quantifying proteins
• establishing protein size

Question 7

Why do proteins in SDS-PAGE move towards the cathode?

Answer 7

negatively charged by surrounding SDS molecules

Question 8

2 methods to identify an unknown protein following SDS page?

Answer 8

1) Western Blotting (immunoblotting)

2) Mass Spectrometry

Question 9

Explain process of western blotting? Which molecules are used in each step? Different results possible?

Answer 9

Nitrocellulose paper placed against gel
-> electric current is applied to the gel so that the separated proteins transfer through the gel and onto the paper in the same pattern as they separated on the SDS-PAGE gel

Incubate Nitrocellulose with BSA as blocking buffer
-> prevents non-specific antibody binding

Add primary antibody to paper which will bind to proteins

Add secondary antibody with conjugate reporter molecule (e.g. Horseradish peroxidase or alkaline phosphatase)
-> binds to primary several-fold -> signal amplification

Reporter molecules detected
HRP by chemiluminescent detection
AP by Colorimetric detection

Question 10

What is chemiluminescent detection? Colorimetric?

Answer 10

Chemiluminescent detection
-> enzyme action detected by film exposure - very sensitive

Colorimetric detection
-> enzyme forms visible coloured precipitate forming bands - cheap, limited sensitivity