Biomolecular techniques (part A)

Question 1

Tissue homogenisation

Answer 1

Waring blender:

• Glass container on top
• Blades process the sample
• Tissue is disrupted

Potter-Elvehjem homogeniser:

• Glass tubes that hold 10-20ml vol.
• Pestle and mortar mechanism
• liquid is separated

Rotor-stator homogeniser:

• Motor driven device
• Blades disrupt structure

Osmotic lysis:

• Blood cells put into highly purified water
• Cells turn into turgid state
• Cells burst
• Only for animal cells

Question 2

Avoiding sample degradation

Answer 2

Key: using a low temperature to maintain cell function for longer.

Adding various buffer that can inhibit endogenous proteases, usually contained in lysosymes- protease inhibitors.

Working quickly, avoid leaving sample at the critical process.

Addition of beneficial ions to the isolation medium eg Na, K, Ca, Mg or Fe can increase stability of dissolved proteins.

Chelating agent: EDTA can remove harmful metal ions.

Addition of reducing agents e.g DTT that will help prevent oxidation of sulphydryl (SH) groups.

Question 3

Purifying samples

Answer 3

Centrifuges (4 types)
Bench top, bench top ultra, refrigerated, floor standing ultra.

Proparative centrifuges: e.g preparing mitochondria.

• Centrifuge rotors
• angle rotor: goes inside the centrifuge, sample starts centrifugation, bands are formed.
• swing out rotor: load sample, centrifugation occurs horizontally, band formation.
• vertical rotor: used for separation of nucleic acids, tubes sit vertically and centrifuged, vertical bands are obtained. when at rest bands revert to horizontal form.

Can be used for quantitative analysis.

Question 4

Ultra centrifuge

Answer 4

Difference between normal and ultra is the speed at which they function.

Ultracentrifuges function a lot faster

Question 5

Differential centrifugation

Answer 5

• Homogenize sample
• sample placed in centrifuged
• spin at 600g for 10 mins, a pellet containing the nuclear fraction is obtained and supernatent.
• spin supernatent at 10,000g, a pellet is obtained containing mitochondria and lysosomes. Extract supernatant
• spin supernatant 40,000g for 1 hour, a pelle containing microsomal fraction, ribosomes, ER is obtained. In addition with soluble enzymes.

Question 6

Density gradient centrifugation

Answer 6

Further purifies samples.

• Create a sucrose gradient with most dense sucrose solution at the bottom and least at the top
• Sample is placed on top of the gradient
• Centrifuged in a swing out motor
• Bands are formed where the position of the sucrose sample MATCHES the density of the sample.

This is called Zonal centrifugation.

Sucrose gradient synthesis:

• Machine which produced a decreasing conc. of sucrose. as the sucrose is poured, the concentration if gradually changed.
• Can be manually done by mixing different concentration of sucrose and pipeting.

Question 7

Isopycnic centrifugation

Answer 7

Instead of having a density gradient then adding sample, A uniform (homogenous) mixture is used, the gradient mixture is usually heavy metal salts.

Question 8

RPM and G-force

Answer 8

a = omega^2x
omega = angular velocity= RPM/60 x 2pi
a due to gravity is 9.81

g-force: acceleration/980(constant)