Advanced Scientific Skills Flashcards
Dissociation constant (Kd) equals:
k2/k1
OR
[R][L] /[RL]
A lower Kd represents
Higher affinity of ligand for receptor
Bmax is:
Total concentration of receptors
[R] + [RL*]
Non-specific binding demonstrates:
A linear increase in [RL] with [L]
Radioligand binds to aspects of the experiment such as test tubes
How is radioligand binding measured?
Filter to trap bound radioligands
Estimate bound radioligands using liquid scintillation counter
How to calculate non-specific binding
Addition of significant excess of competitive ligand to displace radioligand
Remaining quantified radioligand binding is non-specific
Scatchard plot equation
[RL][L]= -[RL*]/ Kd + (Bmax/Kd)
Y axis of scatchard plot
Specific Binding/Total Ligand concentration
X Axis of scatchard plot
Specific ligand binding
Why is total ligand concentration used as free ligand conc
As in reality, bound ligand is very small
What produces a curved scatchard plot
Heterogenous binding e.g. cooperative binding
A high-affinity binding site in cooperative binding would produce
A low Kd with a steep gradient graph
A low affinity binding site would produce
A high Kd with shallow gradient graph
Negative co-operation
Kd less than the average Kd
Due to a decrease in affinity
Positive co-operation
Kd more than the overall Kd
Due to an increase in affinity
Hill Equation
log{B/(Bmax-B)} = nlog[L*] - nlogKd
What does n represent in the Hill equation/plot, and how to calculate this?
n is coefficient of how many ligands may bind a single receptor- the gradient of the line
How to calculate Kd from a Hill plot
X intercept is nlogKd
X axis of Hill plot
log Free (nM)
Y axis of Hill plot
log (B/Bmax-B)
What is the IC50 in an indirect competition binding assay
Concentration of unlabelled ligand that inhibits 50% of radioligand binding
What does the Cheng-Prusoff equation represent
Dissociation constant for unlabelled ligand
Ki= IC50/ (1+ [L*]/Kd)
GPCRs and affinity
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
GTPyS or Gpp(NH)p and activation/affinity
Cannot be hydrolysed so subunits cannot reassociate; means ligand affinity is reduced
What does a low IC50 represent
A low Kd so higher affinity
Left shift of competition binding curve
What does a high IC50 represent
A high Kd so lower affinity
Right shift of competition binding curve
Bigger negative log unlabelled indicates
A smaller concentration (to left of graph)
So a left shift indicates increased affinity at lower concs
Accidental errors
Random in occurrence/magnitude
Normally distributed, measured by standard dev
Include measurement errors etc.
Systematic errors
Arise from experimenter/equipment e
E.g. calibration errors
Minimise with calibration standards etc
Positive controls
Demonstrate effect with a known effector
Done concurrently with negative control to assume appropriate protocol followed
Negative controls
Demonstrate the repsonse with no effect
Done concurrently with positive control to assume appropriate protocol followed
Reagent controls
e.g. blank used in spectroscopy
Solvent in which test substance is dissolved on its own
Method controls
Adding a fixed amount of known internal standard to something being measured to assess reproducibility of a procedure
Purpose of student’s t test, and what it represents
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)
Types of t test
Unpaired T Test
- For two independent groups
Paired T Test
- Two non-independent groups
Interpreting T test
T Value compared to t-table
T value lower than critical value proves the null hypothesis
H0 is
Null hypothesis (no significant difference)
ANOVA (Analysis of Variance) + Post Hoc tests
T-test for multiple groups
Post hoc test follows up significantly significant ANOVA result
Bonferroni post-hoc test
P-significant value/number of tests
Tukey’s Honestly Significant Difference post hoc test
Commonly used if ANOVA assumptions are met
Chi Squared Test purpose
Analysis of binary data/discrete variables
Chi squared value
Sum of {(O-E)^2}/E
O is observed value
E is expected value
Null hypothesis (H0) rejected
if greater than probability listed at p=0.05
how to calculate expected value in Chi squared
Total receiving exposure x (Number with outcome/total patients)
Type 1 (alpha) error
Significance test asserts H0 is false, but actually true
Type 2 (beta) error
Significance test asserts H0 is true, but actually false
Calculation of sample size
Requires a power calculation based on probabilities of Type 1 and 2 errors occuring
What is power, typical value
Probability of detecting a true difference with a particular sample size
80-90% is normally considered reasonable
