Assessment 1 Tutorial

BHS006-6
Molecular Pharmacology
Tutorial 1
Dr Noorjahan J Basheer
[email protected]
Assessment 1 (40%)
You will produce a concise and comprehensive report,
(~3000 words) related to the skills and knowledge developed
in the practical that uses
1. Simulating pharmacological experiments to observe effects
of drugs. You will evaluate the drug receptor interaction
2 molecular biology techniques to examine quantitiate changes
in protein profiles in one of the human disease cells.
Assignment hand in date –Friday 24/11/22 – 10am

Practical sessions
Practical 2 Practical 1
• Protein biomarker identification: • Computer simulation: D103
D304 Batch 1:
w45 (Friday, 10th Nov: 10-1pm)
w44 (Friday, 3rd Nov – 10-5pm)
Batch 2:
w45 (Friday, 10th Nov: 2-5pm)
• Attendance to both practicals is compulsory unless approved by

mitigation
• Late comers will not be permitted to attend/submit the practical
• Finish time of the practical may vary from the timetable.
Practical 1
Important:
1. Wear your labcoat and gloves always
2. Don’t touch your phone/device with gloves on
3. Empty tips go into beaker infront of you
4. Gloves and vials go into yellow bin
5. Before you leave today evening, you need to clean
your bench, throw away unwanted stuff (not the gel!)
Practical – 1
Identification of p16 as a biomarker in hypoxia induced
cells
• The aim of this lab based practical session is to utilise an

established protein separation technique (SDS-PAGE) to identify
biomarker (p16), which are present in human cells.
• You will prepare a SDS gel

• Running buffer
• Pre-stained molecular markers (PageRuler)
• Coomassie Blue Stain Solution and destaining solution
• Hypoxia induced and un-induced (control) cells
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: 15µl of the control (10µg total protein) sample.
• Lane 3: 15µl of 24 hour hypoxia induced cell lysate (10µg total
protein)
• 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
Practical 2
Practical 2:
Data analysis of dose-response curves using a
computer software
Agonist Drugs
• Drugs that interact with and activate receptors; they

possess both affinity and efficacy
Two types
– Full – an agonist with maximal efficacy
– Partial – an agonist with less then maximal efficacy
Partial agonists
1. Compounds that can activate receptors but are unable to elicit the
maximal response of the receptor system
2. Similar potency, but lower efficacy: Intrinsic activity = 0~1

Partial agonists
3. Key property of partial agonists: they display both agonistic and

antagonistic effects. In the presence of a full agonist a partial
agonist will act as an antagonist, competing with the full agonist for
the same receptor and thereby reducing the ability of the full agonist
to produce its maximum effect.
4. partial agonists will elicit a very low or no measurable functional

response even when a significant number of receptor are occupied.
Full vs Partial agonists
A full agonist is a drug whose efficacy is sufficient that it produces a

maximal response when less than 100% of receptors are occupied.
A partial agonist has lower efficacy,

such that 100% occupancy elicits only a submaximal response
Antagonist Drug
• Antagonists interact with the receptor but do NOT

change the receptor
• They have affinity but NO efficacy
two types
– Competitive antagonist
– Noncompetitive antagonist
Receptor antagonists
Competitive antagonist
• Reversible or irreversible.
• Bind to the same site as the
endogenous ligand or agonist
• Can be over come by adding more
agonist!
• Their presence produces a right-ward
shift in both the binding and dose-
response curves.
• No change in Emax.
• Similar dose-response curve shapes
A = agonist alone
indicates the presence of a competitive B = antagonist (one concentration)
agonist (competing for the same binding A+B = agonist + antagonist
sites)
Non-competitive antagonist
• Does not prevent formation of the DR complex, but

impairs the conformation change which triggers a response.
• Bind to a site different than the agonist binding site at an
allosteric site.
• Cannot be overcome by adding more agonist
• Emax is reduced but EC50 remains
the same for the unaffected
receptors.
• Dose-response curves will have
different shapes indicating
different binding sites.
Agonists and antagonists (Summary)
• agonist has affinity plus intrinsic activity

• antagonist has affinity but no intrinsic activity
• partial agonist has affinity and less intrinsic activity
• competitive antagonists can be overcome
Practical 2
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
Control Antagonist Antagonist Antagonist Antagonist

concentration (M) concentration (M) concentration (M) concentration (M)
Agonist
Concn of Response Concn of Response Concn of Response Concn of Response Concn of Response
volume
agonist (M) (mm) agonist (M) (mm) agonist (M) (mm) agonist (M) (mm) agonist (M) (mm)
(ml)
Practical 1
These data will enable you to
 draw concentration-response curves

 Calculate EC50 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 EC50 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.
 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
Unable to submit assessment
•Application for mitigating circumstances

• Mitigation team
([email protected])
• Evidence of circumstances required

• If in doubt talk with personal tutor or course leader
• Do so as soon as possible!
Library – 5 th floor 615.1
School librarian: Janine Bhandol [email protected]

Karin Knisely
A Student Handbook for

Writing in Biology
9781319121815
£31.99
John Smith’s University Bookshop

Unit7/9, The Mall
Library resources
https://library.beds.ac.uk/

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BHS006-6

Assignment hand in date –Friday 24/11/22 – 10am

• Attendance to both practicals is compulsory unless approved by

• The aim of this lab based practical session is to utilise an

• You will prepare a SDS gel

• Lane 1: Protein molecular weight Marker 5ul

Stain and De-stain gel(s):

• Remove the gels from the Gel Cassette Sandwich by gently

• Drugs that interact with and activate receptors; they

2. Similar potency, but lower efficacy: Intrinsic activity = 0~1

3. Key property of partial agonists: they display both agonistic and

4. partial agonists will elicit a very low or no measurable functional

A full agonist is a drug whose efficacy is sufficient that it produces a

A partial agonist has lower efficacy,

• Antagonists interact with the receptor but do NOT

• Does not prevent formation of the DR complex, but

• agonist has affinity plus intrinsic activity

 You will use a computing software PCCAL to simulate the effect of

 Add different volumes of Agonist (from 0.002 ml up to 8 ml) that

 In the presence of agonist, add different concentrations of

Control Antagonist Antagonist Antagonist Antagonist

 draw concentration-response curves

 Use the data to draw dose-response curves in Excel.

 Convert agonist concentrations to log10 values to get a sigmoid plot.

 Plot the negative log of the antagonist concentration (x-axis)

 You have to indicate which receptor is involved in the contraction

•Application for mitigating circumstances

• Evidence of circumstances required

School librarian: Janine Bhandol [email protected]

A Student Handbook for

John Smith’s University Bookshop

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