Sample Preparation Flashcards
Plasma
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
Serum
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
Isotonic solutions
Isotonic saline solutions are often used in hospitals
- 140mM Na+
- 5mM K+
- 2.5mM Ca2+
Centrifugation
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
Microcentrifuges
Used to process small volumes of biological molecules (0.5-2.0 mL)
Max speeds = 12,00-14,000 rpm
Cheaper and safer
High-speed centrifuges
Used to process larger volumes of up to several litres
Max speeds = 30,000 rpm
Ultracentrifuges
Used to isolate very small particles such as viruses
Max speeds = 70,000 rpm
Centrifugal rotors
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
Fixed angle rotor
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
Vertical tube rotor
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
Swing bucket rotor
Used for sucrose density gradient centrifugation
Differential centrifugation
Separates particles based on their density
Yields a pellet and supernatant
Density gradient centrifugation
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
Dialysis
The process of separating molecules in a solution by the different in their rates of diffusion through a semipermeable membrane
Principles of dialysis
- A mixture of salt and protein is poured into semipermeable tubing
- The tubing is placed in pure water
- Only the salt will be able to pass through the membrane
- By continually replacing the pure water, eventually all the salt will have diffused out of the semipermeable tubing
- Hence the protein has been purified