BHS006-6 Molecular Pharmacology

BHS006-6
Molecular Pharmacology
Assessment 1
Dr Noorjahan J Basheer
noorjahan.basheer@beds.ac.uk
03-11-2020
Assessment 1 (30%)
You will produce a concise and comprehensive report,
related to the skills and knowledge developed in the
practical that uses
• 1. computing software to generate dose response data
and evaluates the drug receptor interaction;
• 2. applies proteomic methods to examine protein
profiles in one of the human disease cells.
Assignment hand in date –Friday 20/11/20 – 10am
Practical sessions
Practical 1
• Computer simulation
Group 1:
Wednesday – 4th Nov
2-5pm: Room – D201
Group 2:
Friday- 6th Nov
2-5pm: Room – D201
Practical 2
• protein biomarker identification
All students
Friday: 6th Nov
9.30-2pm
Room: D304
Practical 1
Simulation of drug effects on the guinea pig ileum
 You will use a computing software PCCAL to simulate the effect of
an agonist and four different concentrations of an antagonist, on
guinea pig ileum.
 Add different volumes of Agonist (from 0.002 ml up to 8 ml) that
would correspond to various concentrations (M) and note down
the response (in mm)
 In the presence of agonist, add different concentrations of
antagonist. Note down the response
Data analysis on dose response curve

Practical 1
These data will enable you to
 draw concentration-response curves
 Calculate EC
50 values from the dose response curve
 With the help of EC50 values you can also calculate dose ratio
 and draw schild plot
 Schild plot will enable you to find out the pA2 value for the
antagonist
 You have to find out type of antagonism from dose response curve
 You have to indicate which receptor is involved in the contraction
of guinea pig ileum.
What you need for your practical report
 Use the data to draw dose-response curves in Excel.
 Convert agonist concentrations to log10 values to get a sigmoid plot.
Plot the responses (mm) against the agonist concentrations (M) in a semi
log (x) graph.
 In total you need to construct a dose response curve with agonist alone
and in the presence of antagonist – all in one graph
 You will need to determine the EC
50 value for agonist.
 Inverse log EC50 and calculate the dose ratio (DR) using the formula
 Dose ratio formula: EC50 for agonist obtained in the presence of
antagonist (A1, A2, A3, A4)/ EC50 of agonist obtained in the absence of
antagonist (A)
What you need for your practical report
 Plot the negative log of the antagonist concentration (x-axis)
against the log of the (DR-1) in excel. This is called as Schild plot.
You can derive pA2 for antagonist value from the graph.
 Alternatively, you can also derive pA2 for antagonist value from the
following equation.
 Log (DR-1) = log (antagonist concentration) – pA2
 You have to find out type of antagonism from dose response curve
 You have to indicate which receptor is involved in the contraction
of guinea pig ileum.
Example – dose response curve
Example – Schild plot
Practical – 2
Biomarker identification in human breast cancer cells
• The aim of this lab based practical session is to utilise an
established protein separation technique (SDS-PAGE) to identify
possible biomarkers, which are present in 50pM Estradiol treated
human breast cancer cells versus control cells.
• You will use a pre-casted SDS gel
• Running buffer
• Pre-stained molecular markers (PageRuler)
• Coomassie Blue Stain Solution and destaining solution
• Human CML cell line: CAMA-1 cell line
• Drug used to treat CAMA-1: 50pM Estradiol
Procedure
• Heat all samples in a heat block (set to 100C) for 2 mins and then
cool them down to room temperature
• Load each of the samples into the wells using a gel loading tip.
Load samples slowly to allow them to settle. Be careful not to
puncture the bottom of the well with the tip.
• Lane 1: Protein molecular weight Marker 5ul
• Lane 2: Control cell lysate – 20ul
• Lane 3: treated cell lysate -1 – 20ul
• Lane 4: treated cell lysate -2 – 20ul
• Run at 200 volts constant for about 35 mins, until the bromphenol
blue dye-front has migrated 0.5-1 cm to the bottom of gel.
Procedure
Stain and De-stain gel(s):
• Remove the gels from the Gel Cassette Sandwich by gently
separating the two plates of the gel cassette.
• The gel is then submerged with 20-30ml commassie blue for 15
mins with gently shaking.
• Rinse the gel with fast distaining solution for 20 minutes, followed
by weak distaining solution overnight with gentle shaking.
• Rinse the gel with water until the protein bands are seen clearly.
• Use camera or mobile phone to take photo of the gel for
Assessment write-up (or use gel doc)
https://www.bio-rad.com/en-uk/product/mini-protean-tgx-stain-free-precast-gels?ID=N3GRU1E8Z
Pre-stained protein
molecular weight marker: 10 – 170kDa
Unable to submit assessment
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(mitigation@beds.ac.uk)
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• Do so as soon as possible!
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