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Practicum Molecular Biophysics

Protocol for the Week no.3


~Thomas Leonard

by: Katarina Gacevic


Matr. No. 01428558
Example no.1- Measuring protein concentration

In this experiment we have used two different spectroscopic methods to determine the
concentrations of our samples. The two methods that we have used are NanoDrop and
Bradford assay(plate reader).
NanoDrop allows for the fast analysis of 0.5–2.0µl samples, without the need for cuvettes
or capillaries. With the arm open, a sample is pipetted directly onto the pedestal. The pedestal
then moves to automatically adjust for an optimal path length (0.05–1mm). To be able to
measure it properly, our sample does not need to have large volume. The one necessary thing
is that NanoDrop can not measure samples that have very low concentration. In case of this
measurement, the higher the concentration, the better. However, the fault of NanoDrop is that
our sample can be contaminated easily, mostly by non cleaning the pedestal properly.
When it comes to Bradford assay, it is a bit less precise than NanoDrop. It is based on
absorbance shift of Coomassie Brilliant Blue dye. Under acidic conditions, the red form of the
dye is converted into its blue form, binding to the protein being assayed. If there's no protein to
bind, then the solution will remain brown. Also, if there is a protein in our sample, but the said
protein doesn’t have the component (usually enough aromatic residues) necessary to bind the
dye , it will still remain brown and give us false result. The BA allows measuring of multiple
samples at the same time. It is one of the fastest techniques for measuring the concentration of
proteins. However, because of the amount of dilutions needed, there is a big space for error.
Measurement
During this experiment we have measured a protein of MW= 66.5 kg/mol.

a) NanoDrop (measured at 280nm)


We have prepared a dilution series from our stock-solution (8mg/ml) in physiological
buffer. In the table below we can see the exact concentrations of each of our solutions.

C(protein) [mg/ml] C(protein) [mol/l] C(protein) [μM]


8 0.00012 120
4 0.000061 61
2 0.000031 31
1 0.000015 15
0.5 0.0000076 7.6
0.25 0.0000038 3.8
0.125 0.00000191 1.91
0.0625 0.00000095 0.95
0.0312 0.00000047 0.47
0.0156 0.00000024 0.24
We have then measured each of our samples+unknown one 3 times and have gotten the
following results.
C(protein) Absorbance 1 Absorbance2 Absorbance3 Mean Standard
[mg/ml] deviation
8 0.558 0.564 0.558 0.56 0.003464
4 0.274 0.274 0.271 0.273 0.001732
2 0.144 0.140 0.142 0.142 0.002
1 0.076 0.075 0.076 0.075666667 0.000577
0.5 0.038 0.040 0.038 0.038666667 0.001155
0.25 0.019 0.021 0.019 0.019666667 0.001155
0.125 0.010 0.010 0.010 0.010 0
0.0625 0.004 0.004 0.006 0.004666667 0.001155
0.0312 0.003 0.001 0.001 0.001666667 0.001155
0.0156 0.001 0.001 0.000 0.000666667 0.000577
Unknown 0.038 0.038 0.039 0.038333333 0.000577

The results were analized in RStudio

Slope: 4.622e-03/μM
Adjustedsted R^2 : 0.9994

Predicted concentration :
c=7.86μM
(Absorbance=0.038);
which would mean that our
Mass concentration: Cm=c*M
Cm=0.5148 g/l

Based on the fact that value of ε is equal to the slope of our linear model, we can conclude that
our ε=4.622 *10^(-3) [1/(μM*cm)]
b)Bradford Assay
For the Bradford Assay we have used 250μl of 1x Bradford reagent and 5 μl of protein
solution. We have pipeted our protein tripplet samples directly into a 96-well microplate. Then
we added 250 μl of the Bradford regent into each dilution and measured the absorbance of
each well at 595nm.

C(Protein) [mg/ml] Absorbance1 Absorbance2 Absorbance3 Mean Standard deviation


8 1.3114 1.3263 1.301 1.3129 0.012717
4 1.2272 1.2085 1.204 1.213233333 0.012303
2 1.0364 1.0629 1.0588 1.0527 0.014264
1 0.8964 0.8475 0.8519 0.865266667 0.027052
0.5 0.6582 0.6857 0.6607 0.6682 0.015207
0.25 0.5509 0.5392 0.5375 0.542533333 0.007295
0.125 0.4687 0.4667 0.4649 0.466766667 0.001901
0.063 0.432 0.4401 0.4475 0.439866667 0.007753
0.032 0.4254 0.4172 0.4231 0.4219 0.00423
0.016 0.4256 0.4199 0.4268 0.4241 0.003686
Blank 0.3887 0.4001 0.3967 0.395166667 0.005853
Unknown 0.6988 0.7465 0.701 0.715433333 0.026927

Slope=0.44215(=ε)
Adjusted R^2 of our linear model :
0.9929
Predicted c in the well= 1.58 μM; and
since we had 1:5 dilution,
c(in sample)=7.9 μM
Mass concentration: 0.52535 g/l

Conclusion:
We can notice that we have gotten fairly similar results with difference of 0.01055. We can
then conclude that our samples were relatively clean and that they were properly measured.
This small difference comes from the fact that we used 2 different methods, and it’s natural to
have at least a small deviation is results.
Example no.2- Screening Protein Stability

In this example we have used a thermal shift assay to analyze the effect of different
factors (like pH, ligand concentration) on protein stability.
We were provided with a protein stock of human Cdc42 and following a pipetting scheme
from above we measured the fluorescence intensity by increasing the temperature slowly, 1
degree at the time.
1 2 3 4 5 6 7 8 9 10 11 12
A pH=4.0 pH=5.0 pH=6.0 pH=7.0
5μl GDP/salt 5μl GDP/salt 5μl GDP/salt 5μl GDP/salt
5μl buffer 5μl buffer 5μl buffer 5μl buffer
10μl H2O 10μl H2O 10μl H2O 10μl H2O

B pH=8.0 pH=9.0 pH=10.0


5μl GDP/salt 5μl GDP/salt 5μl GDP/salt
5μl buffer 5μl buffer 5μl buffer
10μl H2O 10μl H2O 10μl H2O

C
D Apo GDP MgCl GDP/MgCl
5μl buffer 5μl GDP 5μl MgCl 5 μl GDP
10μl H2O 5μl buffer 5μl buffer 5μl MgCl
10μl H2O 10μl H2O 5μl buffer
10μl H2O
E EDTA
5μl EDTA
5μl buffer
10μl H2O
F
G
H
Conclusion:
The part of the graphs above that come before the peek signify that at that temperature,
our protein is still folded. We can notice the rise in the graph when the protein starts unfolding,
and when it comes to the max value, it means that it is completely unfolded. The decrease after
the max is caused by protein aggregation or/and dye dissociation.
Example no.3-Protein characterization- fluorescence polarization/anisotropy

In this example we have used the Perkin Elmer Fluorimeter to measure the fluorescence
anisotropy of mant-ADP in a dilution series of protein Itk kinase (MW= 52 kDa).
Fluorescence anisotropy is the phenomenon where the light emitted by a fluorophore has
unequal intensities along different axes of polarization. It is used to determine the binding
affinity of one molecule to another. It does not depend on the concentration of the
fluorophore, only on its intensity.
In our case, the extinction wavelength was 355nm (slit:12.0nm), emission wavelength was
447nm (slit:6nm), GF=1.33.
First have have started with the highest concentrated protein and added Mant-ADP to
40μM. Then we have measured the 120μl of said mixture.

Concentration(μM) Mean(μM) SD
280 0.204677419 0.005381370
140 0.180903226 0.006155512
70 0.148625000 0.011499649
35 0.123064516 0.006109749
17.5 0.081935484 0.006777096
8.75 0.058935484 0.007496379
4.38 0.044064516 0.005881811
2.19 0.015129032 0.006525549
1.09 0.008193548 0.006564650
0.55 0.004093750 0.007003959
blank 0.029406250 0.011207311
Since our plot was not linear we had to adapt our linear model. I have taken only the first 4
values to be relevant for it, since it’s only in that range that our function grows linearly.
After fitting the model, I have simply used one of the many function R has, and calculated
the slope of our linear model.
The slope is k=-0.038871
From this value we can easily find Kd since slope=1/-Kd

Kd=1/-k
Kd=1/0.038871
Kd=25.7261μM

Conclusion
Kd is equilibrium dissociation constant. It Is used to determine the strength of binding of a
single molecule to it’s ligand. The bigger the Kd, the lower the affinity.
Based on the results of our experiment we can see that anisotropy rises with concentration
of the protein. The bound complex of protein and Mant-ADP grows bigger with more added
protein, meaning that it will tumble slower , keeping the polarization in balance/affecting it
less.
Beside measuring the binding affinity of the molecules, this method can also be used in
microscopy( to study local viscosity of the cytosol or membranes and their composition) . It has
also been known to be used to detect the binding of molecules to their partners in signaling
cascades in response to certain cues.
Example no.4-Protein characterization- diffusion coefficient

In this last example, we have used the Fluorescence correlation spectroscopy to measure
the concentration c and the diffusion coefficient D.
We have measured fluctuations for Atto488 and the fluorescent protein EGFP. After
recording our results we have fitted the curve for both. Also we were able to determine the
number of particles N (average number of fluorophores in the confocal volume), confocal
volume Wxy, diffusion time tD, which are shown in the tables below.(necessary calculations
are shown below the table)

Atto488 EGFP V=0.528 fl= 0.528*10^(-15) l


N 3.8075105 1.5776978333 NA=6.022*10^23 [1/mol]
n[mol] 0.6323* 10^(-23) 0.261989 * 10^(-23)
C[mol/l] 1.197* 10^(-8) 0.49619* 10^(-8)

n= N/NA
V=0.528*10^(-15) l
n(Atto488)= 3.8075105/ 6.022*10^23 [1/mol]
n(Atto488)= 0.6323* 10^(-23) mol

n(EGFP)=1.5776978333/6.022*10^23 [1/mol]
n(EGFP)=0.261989 * 10^(-23) mol

c=n/V
c(Atto488)= 0.6323* 10^(-23) mol/0.528*10^(-15) l
c(Atto488)=1.197* 10^(-8) [mol/l]

c(EGFP)= 0.261989 * 10^(-23)mol/ 0.528*10^(-15) l


c(EGFP)= 0.49619* 10^(-8) mol/l
Diffusion coefficient:

Diffusion time- tD [ms] Atto488 EGFP


#1 0.043610 0.100345 Wxy= 0.2353 μm= 2.353 *10^(-7)m
#2 0.044694 0.092135 η= 0.001 kg*m2/s*s
#3 0.043425 0.098680
#4 0.045764 0.104017 M(Atto488)=804g/mol=0.804 kg/mol
#5 0.042498 0.097059 M(EGFP)= 32.7kg/mol
#6 0.043169 0.097564 kb= 1.38*10^(-23) J/K
Mean [ms] 0.04386 0.0983 T=293.15K
Mean [s] 0.00004386 0.0000983
ρ=1200kg/m3
Dexp= Wxy^2 /4tD

Dexp(Atto488)= (2.353 *10^(-7)m)^2/ 4*0.00004386


Dexp(Atto488)= 3.155* 10^(-10) m^2/s
Dexp(EGFP)= (2.353 *10^(-7)m)^2/ 4* 0.0000983
Dexp(EGFP)= 1.4* 10^(-10) m^2/s

3
rtheo= √3𝑀/4𝑁𝑎𝜋𝜌

rtheo (Atto488)=6.429* 10^(-10) m


rtheo(EGFP)=22.109* 10^(-10) m

D=(kB*T)/(6πηr)
D(Atto488)= 3.3399*10^(-10) m2/s
D(EGFP)= 9.71*10(-11) m2/s
Conclusion:
Based on our measurments, we can see that in both cases our Dexp is higher that the
theoretical D. Dexp is connected to the diffusion time, while theoretical D depends on the
particle number. The shorter the diffusion time, bigger is Dexp.
We can also notice that Diffusion coefficients of EGFP are smaller than D from Atto488.
That could be explained with the fact that EGFP molecule is bigger, hence it needs more time to
diffuse. The difference in theoretical D can be explained by the fact that there are more EGFP
particles in the confocal volume-which directly affects D.