Advanced Scientific Skills

Question 1

Dissociation constant (Kd) equals:

Answer 1

k2/k1
OR

[R][L] /[RL]

Question 2

A lower Kd represents

Answer 2

Higher affinity of ligand for receptor

Question 3

Bmax is:

Answer 3

Total concentration of receptors

[R] + [RL*]

Question 4

Non-specific binding demonstrates:

Answer 4

A linear increase in [RL] with [L]

Radioligand binds to aspects of the experiment such as test tubes

Question 5

How is radioligand binding measured?

Answer 5

Filter to trap bound radioligands

Estimate bound radioligands using liquid scintillation counter

Question 6

How to calculate non-specific binding

Answer 6

Addition of significant excess of competitive ligand to displace radioligand

Remaining quantified radioligand binding is non-specific

Question 7

Scatchard plot equation

Answer 7

[RL][L]= -[RL*]/ Kd + (Bmax/Kd)

Question 8

Y axis of scatchard plot

Answer 8

Specific Binding/Total Ligand concentration

Question 9

X Axis of scatchard plot

Answer 9

Specific ligand binding

Question 10

Why is total ligand concentration used as free ligand conc

Answer 10

As in reality, bound ligand is very small

Question 11

What produces a curved scatchard plot

Answer 11

Heterogenous binding e.g. cooperative binding

Question 12

A high-affinity binding site in cooperative binding would produce

Answer 12

A low Kd with a steep gradient graph

Question 13

A low affinity binding site would produce

Answer 13

A high Kd with shallow gradient graph

Question 14

Negative co-operation

Answer 14

Kd less than the average Kd

Due to a decrease in affinity

Question 15

Positive co-operation

Answer 15

Kd more than the overall Kd

Due to an increase in affinity

Question 16

Hill Equation

Answer 16

log{B/(Bmax-B)} = nlog[L*] - nlogKd

Question 17

What does n represent in the Hill equation/plot, and how to calculate this?

Answer 17

n is coefficient of how many ligands may bind a single receptor- the gradient of the line

Question 18

How to calculate Kd from a Hill plot

Answer 18

X intercept is nlogKd

Question 19

X axis of Hill plot

Answer 19

log Free (nM)

Question 20

Y axis of Hill plot

Answer 20

log (B/Bmax-B)

Question 21

What is the IC50 in an indirect competition binding assay

Answer 21

Concentration of unlabelled ligand that inhibits 50% of radioligand binding

Question 22

What does the Cheng-Prusoff equation represent

Answer 22

Dissociation constant for unlabelled ligand

Ki= IC50/ (1+ [L*]/Kd)

Question 23

GPCRs and affinity

Answer 23

G-protein is coupled with GDP bound (to alpha subunit)
High affinity for Beta-gamma subunit

Activation dissociates alpha from beta-gamma, and GTP replaces GDP

GTP is hydrolysed back into GDP, increasing the alpha subunit affinity for beta-gamma allowing receptor coupling

Question 24

GTPyS or Gpp(NH)p and activation/affinity

Answer 24

Cannot be hydrolysed so subunits cannot reassociate; means ligand affinity is reduced

Question 25

What does a low IC50 represent

Answer 25

A low Kd so higher affinity | Left shift of competition binding curve

Question 26

What does a high IC50 represent

Answer 26

A high Kd so lower affinity | Right shift of competition binding curve

Question 27

Bigger negative log unlabelled indicates

Answer 27

A smaller concentration (to left of graph) So a left shift indicates increased affinity at lower concs

Question 28

Accidental errors

Answer 28

Random in occurrence/magnitude Normally distributed, measured by standard dev Include measurement errors etc.

Question 29

Systematic errors

Answer 29

Arise from experimenter/equipment e E.g. calibration errors Minimise with calibration standards etc

Question 30

Positive controls

Answer 30

Demonstrate effect with a known effector Done concurrently with negative control to assume appropriate protocol followed

Question 31

Negative controls

Answer 31

Demonstrate the repsonse with no effect Done concurrently with positive control to assume appropriate protocol followed

Question 32

Reagent controls

Answer 32

e.g. blank used in spectroscopy | Solvent in which test substance is dissolved on its own

Question 33

Method controls

Answer 33

Adding a fixed amount of known internal standard to something being measured to assess reproducibility of a procedure

Question 34

Purpose of student's t test, and what it represents

Answer 34

Determine if the means of two groups are statistically significant Assumes a normal distribution T value represents ratio of 'signal' (variance between groups) to 'noise' (variance within groups)

Question 35

Types of t test

Answer 35

Unpaired T Test - For two independent groups Paired T Test - Two non-independent groups

Question 36

Interpreting T test

Answer 36

T Value compared to t-table | T value lower than critical value proves the null hypothesis

Question 37

H0 is

Answer 37

Null hypothesis (no significant difference)

Question 38

ANOVA (Analysis of Variance) + Post Hoc tests

Answer 38

T-test for multiple groups | Post hoc test follows up significantly significant ANOVA result

Question 39

Bonferroni post-hoc test

Answer 39

P-significant value/number of tests

Question 40

Tukey's Honestly Significant Difference post hoc test

Answer 40

Commonly used if ANOVA assumptions are met

Question 41

Chi Squared Test purpose

Answer 41

Analysis of binary data/discrete variables

Question 42

Chi squared value

Answer 42

Sum of {(O-E)^2}/E O is observed value E is expected value

Question 43

Null hypothesis (H0) rejected

Answer 43

if greater than probability listed at p=0.05

Question 44

how to calculate expected value in Chi squared

Answer 44

Total receiving exposure x (Number with outcome/total patients)

Question 45

Type 1 (alpha) error

Answer 45

Significance test asserts H0 is false, but actually true

Question 46

Type 2 (beta) error

Answer 46

Significance test asserts H0 is true, but actually false

Question 47

Calculation of sample size

Answer 47

Requires a power calculation based on probabilities of Type 1 and 2 errors occuring

Question 48

What is power, typical value

Answer 48

Probability of detecting a true difference with a particular sample size 80-90% is normally considered reasonable