BM7042 Tutorial 1

1.
a. What law governs the relationship between concentration and
absorbance in spectrometry?

Beer Lambert law

A = .l.c
A – absorbance;  – molar absorbtivity l – path length e.g. 1 cm
c-concentration

b. What is the key property of an analyte that determines the sensitivity of

direct detection by spectrophotometry?

Molar absorbtivity; caused by organic molecules containing double bonds e.g.

beta-carotene; haem and breakdown products such as bilirubin; heavy metals
such as Cu2+, Fe2+ and Ni2+

4 marks
2. Describe two uses of electrophoresis to analyse proteins in a sample.

Identification of protein isoforms e.g. enzyme isoforms such as lactate

dehydrogenase (LDH), creatine kinase (tissue damage), haemoglobin – sickle
cell anemia

Serum protein analysis – detecting multiple myeloma (monoclonal IgG); see

slides in lecture

Western blotting – detect presence of specific protein in a sample e.g HIV

membrane proteins in blood

10 marks
3. Name two analytical techniques involving the use of antibodies and
describe key features as indicated:
a. Technique;
b. Sample format;
c. Detection method.
d. Is it quantitative?
e. What key piece of equipment is required?
20 marks
a) ELISA
b) Soluble protein extract from tissue; serum - suitable for application to
a 96 well plate
c) Use secondary AB labelled with HRP – colourimetric substrate or a
fluorescent or a luminescent substrate (latter two are more sensitive)
d) Yes – need standard ie. known concentration of target antigen
(standard curve)
e) Plate reader

a) Flow cytometry
b) Cell analysis – white blood cells e.g CD4+ T-cells in HIV diagnosis
c) Antibodies – detected by lasers which excite fluorescent label on the
Ab
d) Yes – can give a count of cells which are CD4+ in the sample population
e.g cell concentration / count / % of total cells
e) Flow cytometer
4. There are several variations of PCR. List two variants and indicate
a. The name;
b. The application;
c. The type of sample that can be analysed;
d. Briefly, how the variant differs from “classical” PCR.
20 marks
qPCR quantitative (real time)
a) to quantify target copy number (concentration)
b) DNA extracted from blood or tissue
c) Faster, measures amplicon concentration in real time,
quantitative over a broad range, 10 4 (classic PCR 10), very
sensitive

Reverse transcriptase PCR

a) Detection / quantification of mRNA
b) Cell lysate – extract total RNA
c) Allows detection of RNA – after conversion to DNA.