Sample Preparation

Question 1

Plasma

Answer 1

Blood contains 55% plasma

Plasma contains water, proteins, glucose, clotting factors, electrolytes and hormones

Plasma is obtained by centrifuging a tube of blood containing an anticoagulant

Plasma will have a buffy coat

Question 2

Serum

Answer 2

Serum has the same components of plasma

However in this case, the plasma has been allowed to coagulate

Serum can be obtained by centrifuging a tube of blood without an anticoagulant

Question 3

Isotonic solutions

Answer 3

Isotonic saline solutions are often used in hospitals

• 140mM Na+
• 5mM K+
• 2.5mM Ca2+

Question 4

Centrifugation

Answer 4

The application of a centrifugal force for the separation or sedimentation of heterogenous mixtures with a centrifuge

Cellular components can be separated based on their density

A vacuum must also be used to prevent rapid overheating of the sample

Question 5

Microcentrifuges

Answer 5

Used to process small volumes of biological molecules (0.5-2.0 mL)

Max speeds = 12,00-14,000 rpm

Cheaper and safer

Question 6

High-speed centrifuges

Answer 6

Used to process larger volumes of up to several litres

Max speeds = 30,000 rpm

Question 7

Ultracentrifuges

Answer 7

Used to isolate very small particles such as viruses

Max speeds = 70,000 rpm

Question 8

Centrifugal rotors

Answer 8

The rotor is the moving part of the centrifuge

It is typically labelled denoting the type of centrifuge it is compatible with

Rotor top speed will be downgraded every 2 years

Question 9

Fixed angle rotor

Answer 9

With fixed angle rotors the sample tube is held at an angle of 14-40 degrees

The centrifugal force is then applied at an angle

Question 10

Vertical tube rotor

Answer 10

With vertical tube rotors the sample tube is held upright

The centrifugal force is then applied to the side of the tube

Useful for separating very small particles

Question 11

Swing bucket rotor

Answer 11

Used for sucrose density gradient centrifugation

Question 12

Differential centrifugation

Answer 12

Separates particles based on their density

Yields a pellet and supernatant

Question 13

Density gradient centrifugation

Answer 13

Also called isopycnic centrifugation

Involves a density gradient being made of sucrose

Particles then equilibrate at their relevant density within the gradient

Caesium chloride can be used to self-generate a gradient

Question 14

Dialysis

Answer 14

The process of separating molecules in a solution by the different in their rates of diffusion through a semipermeable membrane

Question 15

Principles of dialysis

Answer 15

1. A mixture of salt and protein is poured into semipermeable tubing
2. The tubing is placed in pure water
3. Only the salt will be able to pass through the membrane
4. By continually replacing the pure water, eventually all the salt will have diffused out of the semipermeable tubing
5. Hence the protein has been purified

Question 16

Applications of dialysis

Answer 16

• Removal of unwanted molecules
• Buffer exchange
• Drug binding studies