Practicals Flashcards
What is electrophoresis and Spectrophotometry
Electrophoresis - analysing molecules on the basis of charge, by measuring their migration in an electric field.
Spectrophotometry - analysing molecules on the basis of their spectral properties.
Define absorbance
Absorbance is the fraction of incident light absorbed by a solution which is measured.
(log10 - light transmitted through blank solution)
How much light is transmitted through a solution with A=1.0
10% of light
What wavelength is maximum absorption found for haemoglobin and what colour is the light
540nm
Light is green
Why is the opposite colour seen during spectrophotometry
When white light is passed through a coloured solution, certain wavelength of light are selectively absorbed, and the colour of the solution is that of the remaining light which is transmitted.
What does sodium dithionite do to haemoglobin
Removes oxygen
What is Beer-Lambert law
The absorbance of a solution is proportional to the concentration of the absorbing solute, and to the distance travelled by the light through the sample.
What does the sickle cell mutation do to haemoglobin
Single change of the amino acid at position 6
glutamate (-ve) changes to valine (uncharged)
How is HbA and HbS separated in electrophoresis
HbA moves further than HbS as it is more negatively charged
How do you set up a microscope
- Focus the image on the lowest magnification
- Close the field iris
- Focus the condenser until you get a sharp image of the edge of the disc of light 4. Open the field iris until the whole field of view is illuminated
- Adjust the condenser iris - start with it fully open an close it until the image
only just begins to darken
What is the stain used for white blood cell microscopy
haematoxylin and eosin (H&E)
H- purple/blue - acids&DNA
E - pink - protein
What is Hct, MCV, MCH and MCHC
Hct = the percentage of the volume of a sample of blood occupied by the red cells MCV = Mean Corpuscular Volume MCH = Mean Corpuscular Haemoglobin MCHC = Mean Corpuscular Haemoglobin Concentration
How is haematocrit measured
draw up some blood via capillary action
After centrifuging the blood will separate into plasma at the top and tightly packed red and white cells at the bottom
The ratio of the length of the red cell column to the total length of the cells and the plasma .
How is haemoglobin concentration measured in the blood
Release haemoglobin and converting it to cyanmethaemoglobin
What is Vmax
Maximum enzyme velocity
What is Km
The substrate concentration at which half Vmax is achieved
How can Vmax be found
The Y intercept is 1/Vmax
How can Km be found
X intercept is -1/Km
What is the effect on Vmax and Km with inhibitors
competitive - Vmax remains the same, Km increases
non-competitive - Vmax decreases and Km remains the same
What are the axis for the Lineweaver-Burk plot
Y - 1/V
X - 1/S
