Introduction to Statistics

with GraphPad Prism 7

Outline of the course

• Power analysis with G*Power

• Basic structure of a GraphPad Prism project

• Analysis of qualitative data

• Chi-square test

• Analysis of quantitative data

• t-test, ANOVA, correlation and curve fitting
Power analysis
• Definition of power: probability that a statistical test will reject
a false null hypothesis (H0) when the alternative hypothesis (H1) is
true.
• Translation: statistical power is the likelihood that a test will
detect an effect when there is an effect to be detected.

• Main output of a power analysis:

• Estimation of an appropriate sample size
• Too big: waste of resources,
• Too small: may miss the effect (p>0.05)+ waste of resources,
• Grants: justification of sample size,
• Publications: reviewers ask for power calculation evidence,
• Home office: the 3 R: Replacement, Reduction and Refinement.
 Hypothesis

Experimental design
Choice of a Statistical test

Power analysis: Sample size

Experiment(s)

Data exploration

Statistical analysis of the results

Experimental design
Think stats!!
• Translate the hypothesis into statistical questions:
• What type of data?
• What statistical test ?
• What sample size?

• Very important: Difference between technical and biological replicates.

Technical Biological

n=1 n=3
Power Analysis
The power analysis depends on the relationship
between 6 variables:

• the difference of biological interest

Effect size
• the standard deviation
• the significance level
• the desired power of the experiment
• the sample size
• the alternative hypothesis (ie one or two-sided test)
1 The difference of biological interest
• This is to be determined scientifically, not statistically.
• minimum meaningful effect of biological relevance
• the larger the effect size, the smaller the experiment will
need to be to detect it.

• How to determine it?

• Substantive knowledge, previous research, pilot study …

2 The Standard Deviation (SD)

• Variability of the data
• How to determine it?
• Substantive knowledge, previous research, pilot study …

• In ‘power context’: effect size: combination of both:

• e.g.: Cohen’s d = (Mean 1 – Mean 2)/Pooled SD
3 The significance level
• usually 5% (p<0.05)
•p-value is the probability that a difference as big as the one
observed could be found even if there is no effect.

• Don’t throw away a p-value=0.051 !

4 The desired power of the experiment: 80%

5 The sample size: That’s (often) the all point!

6 The alternative:
• One or two-sided test?
• Fix any five of the variables and a
mathematical relationship can be used to
estimate the sixth.
e.g. What sample size do I need to have a 80% probability (power) to
detect this particular effect (difference and standard deviation) at a
5% significance level using a 2-sided test?

Difference Standard deviation

Sample size

Significance level Power 2-sided test ( )

• Good news:
there are packages that can do the power analysis for
you ... providing you have some prior knowledge of the
key parameters!
difference + standard deviation = effect size

• Free packages:
• G*Power and InVivoStat
• Russ Lenth's power and sample-size page:
• http://www.divms.uiowa.edu/~rlenth/Power/

• Cheap package: StatMate (~ £30)

• Not so cheap package: MedCalc (~ £275)

 Qualitative data

• = not numerical
• = values taken = usually names (also nominal)
• e.g. causes of death in hospital
• Values can be numbers but not numerical
• e.g. group number = numerical label but not unit of
measurement
• Qualitative variable with intrinsic order in their
categories = ordinal
• Particular case: qualitative variable with 2 categories:
binary or dichotomous
• e.g. alive/dead or male/female
 Analysis of qualitative data
Example of data (cats and dogs.xlsx):
• Cats and dogs trained to line dance
• 2 different rewards: food or affection
• Is there a difference between the rewards?

• Is there a significant relationship between the 2 variables?

– are the animals rewarded by food more likely to line dance
than the one rewarded by affection?

• To answer this question: Food Affection

– Contingency table Dance ? ?
– Fisher’s exact test No dance ? ?

But first: how large do your samples need to be?

G*Power
A priori Power Analysis
Example case:
Preliminary results from a pilot study on cats:
25% line-danced after having received affection
as a reward vs. 70% after having received food.

Power analysis with G*Power = 4 steps

Step1: choice of Test family

G*Power

Step 2 : choice of Statistical test

Fisher’s exact test or Chi-square for 2x2 tables

G*Power

Step 3: Type of power analysis

G*Power

Step 4: Choice of Parameters

Tricky bit: need information on the size of the
difference and the variability.
G*Power
Output:
If the values from the pilot study are good predictors and
if you use a sample of n=23 for each group,
you will achieve a power of 81%.
The Null hypothesis and the error types

• The null hypothesis (H0): H0 = no effect

– e.g.: the animals rewarded by food are as likely to line dance as the one
rewarded by affection
• The aim of a statistical test is to reject or not H0.

Statistical decision True state of H0

H0 True (no effect) H0 False (effect)
Reject H0 Type I error α Correct
False Positive True Positive
Do not reject H0 Correct Type II error β
True Negative False Negative

• Traditionally, a test or a difference are said to be

“significant” if the probability of type I error is: α =< 0.05
• High specificity = low False Positives = low Type I error
• High sensitivity = low False Negatives = low Type II error
 Chi-square test
• In a chi-square test, the observed frequencies for two or
more groups are compared with expected frequencies
by chance.

– With observed frequency = collected data

• Example with the cats and dogs.xlsx

 Chi-square test (2)
Did they dance? * Type of Traini ng * Animal Crosstabul ation
Example: expected frequency of cats line
Ty pe of Training dancing after having received food as a
Animal
Food as
Reward
Af f ect ion as
Reward Total reward:
Cat Did they Y es Count 26 6 32
dance?
Direct counts approach:
% wit hin Did they dance? 81.3% 18.8% 100.0%
No Count 6 30 36
% wit hin Did they dance? 16.7% 83.3% 100.0%
Total Count 32 36 68 Expected frequency=(row total)*(column total)/grand total
% wit hin Did they dance? 47.1% 52.9% 100.0% = 32*32/68 = 15.1
Dog Did they Y es Count 23 24 47
dance?
Probability approach:
% wit hin Did they dance? 48.9% 51.1% 100.0%
No Count 9 10 19
% wit hin Did they dance? 47.4% 52.6% 100.0%
Total Count 32 34 66 Probability of line dancing: 32/68
% wit hin Did they dance? 48.5% 51.5% 100.0%
Probability of receiving food: 32/68

Expected frequency:(32/68)*(32/68)=0.22
Did they dance? * Type of Training * Animal Crosstabulati on

Ty pe of Training
22% of 68 = 15.1
Food as Af f ect ion as
Animal Reward Reward Total
Cat Did they Y es Count 26 6 32
dance? Expected Count 15.1 16.9 32.0

For the cats:

No Count 6 30 36
Expected Count 16.9 19.1 36.0
Total Count 32 36 68

Chi2 = (26-15.1)2/15.1 + (6-16.9)2/16.9 +

Expected Count 32.0 36.0 68.0
Dog Did they Y es Count 23 24 47

(6-16.9)2 /16.9 + (30-19.1)2/19.1 = 28.4

dance? Expected Count 22.8 24.2 47.0
No Count 9 10 19
Expected Count 9.2 9.8 19.0

Is 28.4 big enough

Total Count 32 34 66
Expected Count 32.0 34.0 66.0

for the test to be significant?

 Chi-square test: results

Dog Cat
30
D a3n0c e Y e s D ance Y es
D ance N o D ance N o

20 20
C o u n ts

C o u n ts
10 10

0 0
Food A f f e c t io n Food A f f e c t io n

• In our example:
cats are more likely to line dance if they are given food as
reward than affection (p<0.0001) whereas dogs don’t mind
(p>0.99).
 Quantitative data
• They take numerical values (units of measurement)

• Discrete: obtained by counting

– Example: number of students in a class
– values vary by finite specific steps
• or continuous: obtained by measuring
– Example: height of students in a class
– any values

• They can be described by a series of parameters:

– Mean, variance, standard deviation, standard
error and confidence interval
 The mean
• Definition: average of all values in a column
• It can be considered as a model because it
summaries the data
– Example: a group of 5 persons: number of
friends of each members of the group: 1, 2, 3, 3
and 4
• Mean: (1+2+3+3+4)/5 = 2.6 friends per person
– Clearly an hypothetical value

• How can we know that it is an accurate

model?
– Difference between the real data and the model
created
 The mean (2)
• Calculate the magnitude of the differences between
each data and the mean:

From Field, 2000

– Total error = sum of difference

=0
• No errors !
– Positive and negative: they cancel each other out.
 Sum of Squared errors (SS)
• To avoid the problem of the direction of the error: we
square them
– Instead of sum of errors: sum of squared errors (SS):

• SS gives a good measure of the accuracy of the model

• But: dependent upon the amount of data: the more data, the
higher the SS.
• Solution: to divide the SS by the number of observations (N)
• As we are interested in measuring the error in the sample to
estimate the one in the population we divide the SS by N-1
instead of N and we get the variance (S2) = SS/N-1
Variance and standard deviation

• Problem with variance: measure in squared units

– For more convenience, the square root of the variance is
taken to obtain a measure in the same unit as the original
measure:
• the standard deviation
– S.D. = √(SS/N-1) = √(s2) = s

• The standard deviation is a measure of how well the mean

represents the data
 Standard deviation

Small S.D: data close to the mean: Large S.D.: data distant from the mean:
mean is a good fit of the data mean is not an accurate representation
 SD and SEM (SEM = SD/√N)

• What are they about?

– The SD quantifies how much the values vary from

one another: scatter or spread
• The SD does not change predictably as you acquire more
data.

– The SEM quantifies how accurately you know the

true mean of the population.
• Why? Because it takes into account: SD + sample size
• The SEM gets smaller as your sample gets larger
– Why? Because the mean of a large sample is likely to be
closer to the true mean than is the mean of a small sample.
 SD and SEM

The SD quantifies the scatter of the data. The SEM quantifies how much we expect
sample means to vary.
 SD or SEM ?

• If the scatter is caused by biological variability,

it is important to show the variation.
– Report the SD rather than the SEM.
• Better even: show a graph of all data points.

• If you are using an in vitro system with no

biological variability, the scatter is about
experimental imprecision (no biological
meaning).
– Report the SEM to show how well you have
determined the mean.
 Confidence interval

• Range of values that we can be 95% confident contains the true mean of the
population.
- So limits of 95% CI: [Mean - 1.96 SEM; Mean + 1.96 SEM] (SEM = SD/√N)

Error bars Type Description

Standard deviation Descriptive Typical or average difference
between the data points and their
mean.

Standard error Inferential A measure of how variable the

mean will be, if you repeat the
whole study many times.

Confidence interval Inferential A range of values you can be 95%

usually 95% CI confident contains the true mean.
 Analysis of quantitative data

• Choose the correct statistical test to answer

your question:

– They are 2 types of statistical tests:

• Parametric tests with 4 assumptions to be met by the

data,
• Non-parametric tests with no or few assumptions (e.g.
Mann-Whitney test) and/or for qualitative data (e.g.
Fisher’s exact and χ2 tests).
Assumptions of Parametric Data
• All parametric tests have 4 basic assumptions that
must be met for the test to be accurate.
1) Normally distributed data
– Normal shape, bell shape, Gaussian shape

• Transformations can be made to make data suitable

for parametric analysis
 Assumptions of Parametric Data (2)
• Frequent departure from normality:
– Skewness: lack of symmetry of a distribution
Skewness < 0 Skewness = 0 Skewness > 0

– Kurtosis: measure of the degree of ‘peakedness’ in the

distribution
• The two distributions below have the same variance
approximately the same skew, but differ markedly in
kurtosis.

More peaked distribution: kurtosis > 0 Flatter distribution: kurtosis < 0

Assumptions of Parametric Data (3)
2) Homogeneity in variance
• The variance should not change systematically
throughout the data

3) Interval data
• The distance between points of the scale should
be equal at all parts along the scale

4) Independence
• Data from different subjects are independent
– Values corresponding to one subjects do not influence
the values corresponding to another subject.
– Important in repeated measures experiments
 Analysis of quantitative data
• Is there a difference between my groups regarding the
variable I am measuring?
– e.g.: are the mice in the group A heavier than the one in
group B?
• Tests with 2 groups:
– Parametric: t-test
– Non parametric: Mann-Whitney/Wilcoxon rank sum test
• Tests with more than 2 groups:
– Parametric: Analysis of variance (one-way ANOVA)
– Non parametric: Kruskal Wallis

• Is there a relationship between my 2 (continuous)

variables?
– e.g.: is there a relationship between the daily intake in
calories and an increase in body weight?
• Test: Correlation (parametric or non-parametric)
 Remember:
Stats are all about understanding and
controlling variation.

signal If the noise is low then the signal is detectable …

= statistical significance
noise

signal … but if the noise (i.e. interindividual variation) is large

then the same signal will not be detected
noise = no statistical significance

In a statistical test, the ratio of signal to noise

determines the significance.
 Comparison between 2 groups:
t-test
• Basic idea:
– When we are looking at the differences between scores for
2 groups, we have to judge the difference between their
means relative to the spread or variability of their scores
• Ex: comparison of 2 groups control and treatment
t-test (2)
t-test (3)
 SE gap ~ 2 n=3 SE gap ~ 4.5 n=3
13 16

15

Dependent variable
Dependent variable

12
14
11 13
~ 2 x SE: p~0.05 ~ 4.5 x SE: p~0.01
10 12

11
9
10
8 9
A B A B

SE gap ~ 2 n>=10
SE gap ~ 1 n>=10
12.0
11.5
Dependent variable
11.5
Dependent variable

11.0
11.0
~ 1 x SE: p~0.05 ~ 2 x SE: p~0.01
10.5
10.5

10.0 10.0

9.5 9.5
A B A B
 CI overlap ~ 1 n=3 CI overlap ~ 0.5 n=3

14
Dependent variable

Dependent variable
12 15

10 ~ 1 x CI: p~0.05
~ 0.5 x CI: p~0.01
8 10

A B
A B
CI overlap ~ 0.5 n>=10
CI overlap ~ 0 n>=10
12
12
Dependent variable

Dependent variable
11 11
~ 0.5 x CI: p~0.05
~ 0 x CI: p~0.01
10 10

9 9
A B A B
 t-test (4)
• 3 types:
– Independent t-test
• it compares means for two independent
groups of cases.
– Paired t-test
• it looks at the difference between two
variables for a single group:
– the second sample is the same as the first after
some treatment has been applied
– One-Sample t-test
• it tests whether the mean of a single variable
differs from a specified constant (often 0)
Example: coyote.xlsx

• Question: is there a difference in size between

males and females coyotes?

• 1 Power Analysis
• 2 Plot the data
• 3 Check the assumptions for parametric test
• 4 Statistical analysis: Independent t-test
G*Power
Independent t-test

A priori Power analysis

Example case:

You don’t have data from a

pilot study but you have
found some information in
the literature.

In a study run in similar

conditions as in the one you
intend to run, male coyotes
were found to measure:
92cm+/- 7cm (SD)

You expect a 5% difference

between genders with a
similar variability in the
female sample.

You need a sample size of n=76 (2*38)

 Coyote
110
Maximum

100
Upper Quartile (Q3) 75th percentile

Interquartile Range (IQR)

Length (cm)

90

Median Lower Quartile (Q1) 25th percentile

80

Smallest data value Cutoff = Q1 – 1.5*IQR

> lower cutoff

70 Outlier

60
Male Female
 Assumptions for parametric tests
Histogram of Coyote:Freq. dist. (histogram)
10 Counts OK here Female
but if several groups of different sizes, Male
8
go for percentages
6
Counts

0 707274767880828486889092949698100
102
104
106 707274767880828486889092949698100
102
104
106

15 Bin Center
Female
Male

10
Counts

Normality 
0 69 72 75 78 81 84 87 90 93 96 99 102105 69 72 75 78 81 84 87 90 93 96 99 102105

Bin Center
15
Female
Male

10
Counts

0 68 72 76 80 84 88 92 96 100 104 108 68 72 76 80 84 88 92 96 100 104 108

Bin Center
 100
Independent t-test: example
95 Standard error coyote.xlsx
B ody M ass

90

C o y o te s
85
110

108
80
106
F e m a le M a le
104

102

100
Standard deviation 100

98

96
95 94
B ody M ass

92
90 90

L e n g th (c m )
88
85
86

84
80
82
F e m a le M a le
80

95 78

94 76

93 74
Length (cm)

92 72

91 70

90 68

89 66

88 64

87 62

86 Confidence interval 60
F e m a le M a le
85
Male Female
 Independent t-test: results
coyote.xlsx

Males tend to be longer than females

but not significantly so (p=0.1045).

Homogeneity in variance 

What about the power of the analysis?

Power analysis
You would need a sample 3 times bigger
to reach the accepted power of 80%.

But is a 2.3 cm difference between genders biologically relevant (<3%) ?

Another example of t-test: working memory.xlsx

 The sample size: the bigger the better?

• It takes huge samples to detect tiny differences but tiny samples

to detect huge differences.

• What if the tiny difference is

meaningless?
• Beware of overpower
• Nothing wrong with the stats: it is
all about interpretation of the
results of the test.

• Remember the important first step of

power analysis
• What is the effect size of
biological interest?
working memory.xlsx

Normality 
 Paired t-test: Results
working memory.xlsx

No test for homogeneity of variances

Comparison of more than 2 means

• Why can’t we do several t-tests?

– Because it increases the familywise error
rate.

• What is the familywise error rate?

– The error rate across tests conducted on
the same experimental data.
 Familywise error rate
• Example: if you want to compare 3 groups and you carry out 3 t-
tests, each with a 5% level of significance
• The probability of not making the type I error is 95% (=1 – 0.05)
– the overall probability of no type I errors is:
0.95 * 0.95 * 0.95 = 0.857
– So the probability of making at least one type I error is
1-0.857 = 0.143 or 14.3%
– The probability has increased from 5% to 14.3% !
– If you compare 5 groups instead of 3, the familywise error rate is
40% !!!!! (=1-(0.95)n)

• Solution for multiple comparisons: Analysis of variance

 Analysis of variance
• Extension of the 2 groups comparison of a t-test but
with a slightly different logic:

• t-test = mean1 – mean2

Pooled SD Pooled SD

• ANOVA = variance between means

Pooled SD Pooled SD

• ANOVA compares variances: If variance between the several

means > variance within the groups (random error)
• then the means must be more spread out than it would have been by
chance.
 Analysis of variance
• The statistic for ANOVA is the F ratio.

Variance between the groups

• F=
Variance within the groups (individual variability)

Variation explained by the model (= systematic)

• F=
Variation explained by unsystematic factors (= random variation)

• If the variance amongst sample means is greater

than the error/random variance, then F>1

– In an ANOVA, you test whether F is significantly higher

than 1 or not.
 Analysis of variance
Source of variation Sum of Squares df Mean Square F p-value

Between Groups 2.665 4 0.6663 8.423 <0.0001

Within Groups 5.775 73 0.0791
In Power Analysis:
Total 8.44 77
Pooled SD=√MS(Residual)
• Variance (= SS / N-1) is the mean square
– df: degree of freedom with df = N-1
Hypothetical model

Between groups variability

Within groups variability

Total sum of squares
 Example: protein expression.xlsx

• Question: is there a difference in protein

expression between the 5 cell lines?

• 1 Plot the data

• 2 Check the assumptions for parametric test
• 3 Statistical analysis: ANOVA
 10

Protein expression
8

0
A B C D E
Cell groups
10

0
A B C D E
Cell groups
Parametric tests assumptions
 1.5

Protein expression (Log)

1.0

0.5

0.0

-0.5

-1.0
A B C D E

1.5
Protein expression (Log)

1.0

0.5

0.0

-0.5

-1.0
A B C D E
Cell groups
Parametric tests assumptions

Normality 
 Analysis of variance: Post hoc tests

• The ANOVA is an “omnibus” test: it tells you that

there is (or not) a difference between your means
but not exactly which means are significantly
different from which other ones.
– To find out, you need to apply post hoc tests.

– These post hoc tests should only be used when the

ANOVA finds a significant effect.
Analysis of variance
 Analysis of variance
Results

Homogeneity of variance 

F=0.6727/0.08278=8.13

Post hoc tests

 Correlation
• A correlation coefficient is an index number that measures:
– The magnitude and the direction of the relation between 2
variables
– It is designed to range in value between -1 and +1
 Correlation
• Most widely-used correlation coefficient:
– Pearson product-moment correlation coefficient
“r”

• The 2 variables do not have to be measured in the same

units but they have to be proportional (meaning linearly
related)
– Coefficient of determination:
• r is the correlation between X and Y
• r2 is the coefficient of determination:
– It gives you the proportion of variance in Y that can be
explained by X, in percentage.
 Correlation: example
roe deer.xlsx
• Is there a relationship between parasite burden
and body mass in roe deer?
30
M a le

F e m a le
25

B ody M ass
20

15

10
1 .0 1 .5 2 .0 2 .5 3 .0 3 .5
P a r a s it e s b u r d e n
Correlation: example
roe deer.slsx

There is a negative correlation between

parasite load and fitness but this relationship
is only significant for the males
(p=0.0049 vs. females: p=0.2940).
 Curve fitting
• Dose-response curves
– Nonlinear regression
– Dose-response experiments typically use around 5-10 doses of agonist, equally
spaced on a logarithmic scale.
– Y values are responses.

• The aim is often to determine the IC50 or the EC50

– IC50 (I=Inhibition): concentration of an agonist that provokes a response half way
between the maximal (Top) response and the maximally inhibited (Bottom)
response
– EC50 (E=Effective): concentration that gives half-maximal response

Stimulation: Inhibition:
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) Y=Bottom + (Top-Bottom)/(1+10^((X-LogIC50)))
 Curve fitting: example
Inhibition data.xlsx
500
N o in h ib ito r
400 In h ib ito r

300

200

100

0
-1 0 -8 -6 -4 -2
-1 0 0 lo g (A g o n is t ], M

Step by step analysis and considerations:

1- Choose a Fit:
not necessary to normalize
should choose it when values defining 0 and 100 are precise
variable slope better if plenty of data points (variable slope or 4 parameters)

2- Compare different conditions:

Diff in parameters
Diff between conditions for one or more parameters
Constraint vs no constraint
Diff between conditions for one or more parameters

3- Constrain:
depends on your experiment
depends if your data don’t define the top or the bottom of the curve

4- Weights:
important if you have unequal scatter among replicates
 Curve fitting: example
Inhibition data.xlsx
500
N o in h ib ito r
400 In h ib ito r

300

200

100

0
-1 0 -8 -6 -4 -2
-1 0 0 lo g (A g o n is t ], M

Step by step analysis and considerations:

5- Initial values:
defaults usually OK unless the fit looks funny

6- Range:
defaults usually OK unless you are not interested in the x-variable full range (ie time)

7- Output:
summary table presents same results in a … summarized way.

8- Diagnostics:
check for normality (weights) and outliers (but keep them in the analysis)
check Replicates test
 Curve fitting: example
Inhibition data.xlsx
N o n - n o r m a liz e d d a ta 3 p a r a m e te r s
N o n - n o r m a liz e d d a ta 4 p a r a m e te r s
500
500

450
450

400
400

350
350

300
300

250

R esponse
250
R esponse

200 EC50
200 EC50

150
150 N o in h ib ito r
N o in h ib ito r
100
100 In h ib ito r
In h ib ito r
50
50

0
0
-9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0
-9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0
-5 0 lo g (A g o n is t)
-5 0 lo g (A g o n is t)
-1 0 0
-1 0 0

N o r m a liz e d d a ta 3 p a r a m e te r s
N o r m a liz e d d a ta 4 p a r a m e te r s
110
110
100
100
90
90

80
80

70
70
R e s p o n s e (% )

60
60
EC50
50
50

N o in h ib ito r 40
40

In h ib ito r 30
30 N o in h ib ito r
20
20 In h ib ito r

10
10

0 0
-1 0 . 0 -9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0 -1 0 . 0 -9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0

lo g (A g o n is t) lo g (A g o n is t)
 Curve fitting: example
Inhibition data.xlsx
500
N o n - n o r m a liz e d d a ta 4 p a r a m e te r s
No inhibitor Inhibitor
450

No inhibitor Inhibitor 400

350

300

Replicates test for lack of fit 250

R esponse
SD replicates 22.71 25.52 200 EC50

150 N o in h ib ito r
SD lack of fit 41.84 32.38 100 In h ib ito r
Discrepancy (F) 3.393 1.610 50

-7.158 -6.011
P value 0.0247 0.1989 0
-9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0

Evidence of inadequate model? Yes No lo g (A g o n is t)

-5 0

-1 0 0

N o n - n o r m a liz e d d a ta 3 p a r a m e te r s
500

450

400

Replicates test for lack of fit 350

SD replicates 22.71 25.52 300

SD lack of fit 39.22 30.61 250

R esponse
EC50
Discrepancy (F) 2.982 1.438 200

150

P value 0.0334 0.2478 100

N o in h ib ito r

Evidence of inadequate model? Yes No 50

In h ib ito r
-7.159 -6.017
0
-9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0

-5 0 lo g (A g o n is t)

-1 0 0

N o r m a liz e d d a ta 4 p a r a m e te r s
110

100

Replicates test for lack of fit 90

SD replicates 5.755 7.100 80

70

SD lack of fit 11.00 8.379

R e s p o n s e (% )
60
EC50

Discrepancy (F) 3.656 1.393 50

N o in h ib ito r
40

P value 0.0125 0.2618 30

In h ib ito r

Evidence of inadequate model? Yes No 20

10

0
-7.017 -5.943
-1 0 . 0 -9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0

lo g (A g o n is t)

N o r m a liz e d d a ta 3 p a r a m e te r s
110

100

90

Replicates test for lack of fit 80

SD replicates 5.755 7.100 70

60

SD lack of fit 12.28 9.649 50

Discrepancy (F) 4.553 1.847 40

30
N o in h ib ito r
P value 0.0036 0.1246 20
In h ib ito r

Evidence of inadequate model? Yes No 10

0
-7.031 -5.956
-1 0 . 0 -9 . 5 -9 . 0 -8 . 5 -8 . 0 -7 . 5 -7 . 0 -6 . 5 -6 . 0 -5 . 5 -5 . 0 -4 . 5 -4 . 0 -3 . 5 -3 . 0

lo g (A g o n is t)
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