This document describes an experiment that performed spectrophotometry on citrate-gold nanoparticles (C-AuNPs) of varying sizes to analyze how size influences optical properties. C-AuNPs with diameters of 5, 10, 30, and 60 nm were serially diluted and measured. Peak wavelengths of 520 nm, 530 nm, 620 nm, and 630 nm were observed, suggesting these correspond to particle sizes from smallest to largest. The extinction coefficient of samples containing estimated 10 nm particles was calculated as 0.2152 M-1cm-1 from absorbance versus concentration data. The results demonstrate how size affects the optical properties of nanoparticles.

1. 1
Lab 9: Mammalian Cell Culture
Jacob Feste
010617389
4/13/15
Objective
The objective of this experiment is to learn and perform mammalian cell culture techniques
including cell passage, cell counting, and culture maintenance. This experiment utilizes NIH-3T3
mammalian cells with Trypsin for a dissociation agent during cell passaging. Cell counting will be
performed using a hemocytometer with Trypan blue solution for staining. It is also requires
strict sterilization techniques to be followed, such as the use of ethanol and chemical hoods to
reduce the chances of contamination for sensitive cells such as mammalian cells. NIH-3T3 cells
are mammalian mouse embryonic fibroblast cells from a continuous cell line. Mammalian cells
typically proliferate or divide until there is no longer room for them to do so, and are
considered to be at confluency. Upon the removal of cells the remaining cells will divide, again
until there is no longer room. Cell passaging is the process of removing cells from a culture and
into a sub culture, giving way to cell lines. For this experiment, Trypsin will be used as a
dissociation agent due to its ability to dissociate the anchor the cells make with the surface to
allow removal. NIH-3T3 cells are not altered or damaged by Trypsin, making it ideal to use as a
dissociation agent for these cells. Cell lines utilize this property to “harvest” cells from a culture
of cells with this property, allowing them to be used over and over. NIH-3T3 cells have
genetically been modified to induce immortality for the cells as long as they remain
uncontaminated, allowing them to infinitely be harvested over time and therefore classifying
the NIH-3T3 cell line as continuous. These properties make mammalian cells such as NIH-3T3
and cells from other continuous cell lines desirable for mammalian cell experiments. A
hemocytometer will be used with Trypan blue solution to count the cells after cell passage. A
hemocytometer uses square wells to be filled with culture cells, diluted with Trypan blue for
this experiment, and microscopy to allow visual counting of cells with specific guidelines
underlying which cells are viable to count. Trypan blue is mixed with the cells for a 1:1 dilution
(dilution factor of 2) to stain dead cells blue, indicating for them not to be counted. The
concentration of cells in a volume of culture is given by a formula factoring in cells counted and
the dilution factor. Around a million NIH-3T3 cells are kept in a T25 flask with a 5ml working
volume for cells to reside and grow in, giving around 200,000 cells per ml of suspension.
Therefore, this experiment can be expected to indicate a measured 100,000 cells per ml after a
1:1 Trypan blue dilution and cell counting using the hemocyctometer. Also, since four grids with
a total volume of 4*10-4 ml will be used with a dilution factor of two, we can expect to count a
total of around 20 cells using the hemocytometer. Higher values will simply indicate

2. 2
measurement error while lower values will likely be obtained and due to cells dying by
contamination or with time.
Materials
1. 440 ml of DMEM basic media without L-glutamine with high glucose (cell culture media)
2. 50 ml fetal bovine serum (FBS) (cell culture media)
3. 5 ml Penstrep (penicillin streptomycin) (cell culture media)
4. 5 ml L-glutamine (cell culture media)
5. T25 cell culture flasks
6. NIH-3T3 cells
7. Tissue culture hood
8. Aspirating Pipette
9. Vacuum flask
10. 5 ml transfer pipettes
11. 3 ml PBS
12. 1 ml Trypsin
13. Ethanol
14. Incubator
15. Centrifuge
16. 15 ml centrifuge tube
17. 100 µl Trypan blue solution
18. Kimwipe
19. Hemocytometer
20. Micropipettes
21. 500 µl 200MOI viral media
Procedures
Passaging cells:
1. Move the T25 cell culture flask containing 3T3 cells into the tissue culture hood
2. Take an aspirating pipette and connect it to vacuum flask
3. Remove the cap and aspirate cell culture media (mixture of materials 1-4) – Make sure
the tissue culture flask is in vertical position and the pipette tip does not touch the
culture area
4. Now add 3 ml PBS to the flask using a 5ml transfer pippette
5. Close the lid and leave the flask in horizontal position for a min
6. Now aspirate PBS using the aspirating pipette

3. 3
7. Now add 1ml Trypsin solution to the flask
8. Close and move the flask to the incubator
9. Incubate for 2-3 min or until the cells detach – Check the flask under microscope for cell
detachment
10. Move the flask back to the culture hood
11. Add 4ml of cell culture media using a 5ml transfer pipette and mix to neutralize trypsin
12. Using a transfer pipette transfer the cell suspension into a 15ml centrifuge tube
13. Centrifuge at 300g for 5min
14. Move the tube back to the hood
15. Aspirate the supernatant taking care not to aspirate the cell pellet
16. Now add 5ml of cell culture media and mix well using the same transfer pipette
17. Now transfer 1ml of cell suspension to a new T25 flask
18. Add 4ml of cell culture media
19. Mix gentle and label the flask with cell line name, passage number, initials, and date
20. Place the flask in incubator in the horizontal position
21. Incubate cells for 3-4days
22. After 3- 4 days incubation, remove the old media and add 5 ml of new cell culture media
23. Cell will need a passage after 7 days
Cell counting using hemocytometer:
1. Mix the cell suspension using a transfer pipette – Push the media out through smallest
possible opening forcing them to form single cell suspension
2. Take 100µL of cell suspension into an Eppendorf tube.
3. Now add 100µL of Trypan blue solution and mix well (1:1 dilution with dilution factor of
2)
4. Hemocytometer preparation: spray the cytometer and cover slip with ethanol and wipe
it with kimwipe. Position the cover slip over the chambers.
5. Now take 10µL of the mix using micro pipette tips and add it to the hemocytometer
wells. To do this, center the cover slip on the hemocytometer, lay the pipette tip as
parallel to the ground as possible, and inject the mixture in the well underneath the
cover slip. Make sure the entire surface of the rectangular grid of the hemocytometer is
covered. It is important not to underfill or overfill the chambers
6. Count viable cells using 10X or 20X magnification under the microscope
7. Count the cells that overlay the grid of the hemocytometer at the corners. If cells are on
the border outlining each square, count only the cells on the top and left border of the
square as shown below.
8. Use the following equation to get cell count per mL
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 / 𝑚𝑙 = ((𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ∗ 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟)/4 ) ∗ 10,000

4. 4
Adenoviral Transfection:
1. Prepare 20 MOI viral media by adding 500µL 200MOI viral media to 5ml cell culture
media in a 15ml centrifuge tube
2. Mix the solution by tilting the tube up and down
3. Move the cell culture flask to the hood
4. Aspirate the culture media using an aspirating pipette
5. Add the 20MOI viral media
6. Transfer the cell culture flask back to the incubator
Results
Number of Cells
Counted
Number of Corners
Measured (0.1 mm3)
Dilution Factor Total Number of Cells
per ml
16 4 2 ~ 80,000
Figure 1: Hemocytometer Results after NIH-3T3 Mammalian Cell Passage
Discussion and Conclusion
The results of the experiment support the predictions. The mammalian cell culture techniques
including cell passage, cell counting, and cell maintenance were successfully performed. The
original T25 flask containing the NIH-3T3 cell suspension had a seeding density of around a
million cells per flask or 40,000 cells per cm2, since T25 flasks have a 25 cm2 culture area. They
also have 5 ml working volume of which the cells reside. Therefore, with 1 ml the original flask’s
cell suspension being passaged into a new T25 flask with the same properties, there were
around 200,000 cells passaged in the 1 ml volume. The working volume was obtained in this
flask by adding 4 ml of cell culture media. This gave a seeding density for the second flask of
200,000 cells per flask, or 8,000 cells per cm2. NIH-3T3 cells have a recommended inoculation
density of 300,000 cells (per 20 cm2 dish). T25 flasks have a slightly larger culture area of 25
cm2, possibly recommending more cells, however it can be generally stated that the new flask
has a much more suitable seeding density than the original flask. Since the recommended
inoculation density (inoculum) indicates the density of cells most suitable for inoculation, better
seeding densities are closer to this value. The original flask’s seeding density of a million cells is
much larger than the recommended 300,000 cells, with the large amount of cells likely taking
up either too much room or being too much for the body to handle. The new T25 flask’s
seeding density of 200,000 cells is much better and more suitable as it is closer to the
recommended value and can be grown to be even closer if desired. The results of the
experiment are illustrated by figure 1. Overall, these results indicate that cell passage was
performed and performed with an acceptable degree of accuracy. The presence of viable cells
counted, as well as the count not exceeding that of which it is theoretically maximized to have,

5. 5
indicates that cell passage occurred. It also indicates that cell maintenance, such as sterilization
with ethanol, was quite successful as well. The expected hemocytometer count of 20 cells was
almost reached, with 16 total cells being counted. While no loss in cells after passage was
perhaps possible, the passage procedure was expected to realistically give some loss due to
cells dying naturally over time. While considered sources of error, contamination and
mishandling were predicted due to the NIH-3T3 mammalian cells high sensitivity to
contaminants and physical harm. These properties support that although around 20% of the
cells were lost, the cell passage procedure can still be considered an overall success. The
hemocytometer equation for the estimated total number of cells per volume (#
cells/ml = [[total number of cells counted*dilution factor]/number of grids or corners
measured]*10,000]) supplies an estimated 80,000 cells per ml for our conditions, illustrated in
figure 1. This is also only a 20% loss from the predicted value of 100,000 cells per ml, with the
loss likely caused by the same factors as those for the loss in cell count from the expected
count. The results conclude success overall as many cells were passaged and counted
successfully. The cell passaging such as the one performed in this experiment is necessary for
mammalian cell cultures for multiple reasons. Mammalian cells have long, sensitive cell cycles
and grow slowly, much slower than bacteria cells. Most mammalian cell types used in research
were able to be genetically altered to last longer than natural or forever as long as certain
conditions such as sanitary conditions are maintained, giving way to the possibility of cell lines.
These cells also have a confluency, growing until they run out of room. Widely used mammalian
cell cultures from cell lines or other established mammalian cell harvesting techniques live long
enough to grow at a faster rate than their death rate. Therefore as long as some of the culture
is transferred to another sub culture before reaching confluency, each culture may
hypothetically last forever in continuous cell lines or a large amount of time for finite lines if
maintained under the proper conditions. These properties illustrate the importance of cell
passaging and its usefulness as a culture tool. The culture media we used included DMEM basic
media, FBS, Penstrep, and L-glutamine. The DMEM basic media has high glucose and was
therefore used to provide energy. FBS is an animal serum to give the 3T3 cells a similar
environment to their natural one, with components that give the cells necessary or desired
nutrients. Penstrep is a form of the antibiotic penicillin and is used to protect against
contamination. Finally, L-glutamine serves as a building block for the cells to grow on. Each
ingredient in media such as this have an impactful purpose and vary dependent on the cell
type. The magnitude of error in this experiment is estimated to be small due to the small loss in
cells that could’ve occurred naturally. Contamination, mishandling, inaccurate measurements,
or other human error could’ve been possible but together were not too impactful on the data.
The use of a hemocytometer, however, gives only an estimation and therefore inherent
accuracy error. Hemocytometers are easy to use, relatively cheap, visibly easy to count, and can
distinguish between living and dead cells with dye; however they also only give an estimation
and have their results skewed largely if miscounted for less dense samples while being difficult
to count accurately for more dense samples. Other methods could’ve been performed to count
such as the use of a culture counters or other automated cell counters that are much more

6. 6
expensive, however give much more accurate results and are easier to use. In conclusion,
however, the results indicate that mammalian cell culture techniques such as cell passage,
counting, and maintenance were successful with little loss of cells. The experiment resulted
similar to predicted while also expanding the knowledge of its participants in the area of
mammalian cell culture.

7. 7
DNA-Guided Self-Assembly and Spectrophotometric Analysis of Nanocomposites
Jacob Feste
University of Arkansas, Biomedical Engineering, jtfeste@email.uark.edu
10/11/2015

8. 8 | P a g e
8
Abstract
This experiment performs spectrophotometry to illustrate the influence that size has on
the optical properties of a nanoparticle. Citrate-gold nanoparticles (C-AuNPs) with diameters of
5, 10, 30, and 60 nm were diluted with ddH2O five times and by a factor of two for each dilution.
There were three samples for each of these dilution factors and for each diameter size. The
results indicated peak wavelengths of 520 nm, 530 nm, 620 nm, and 630 nm. Supported by a
darker shift in the visible color of these solutions, it is suggested that the order of these
wavelengths from smallest to largest represent the order of their diameters similarly (520 nm = 5
nm diameter, 630 nm = 60 nm diameter). The average extinction coefficient (ε) for the samples
estimated to contain the 10 nm diameter C-AuNPs was calculated as 0.2152 (M-1 cm-1) by taking
the slope of absorbance versus concentration.
Nomenclature
A = Absorbance (unit less)
ε = Extinction Coefficient (M-1 cm-1)
C = Concentration (M)
L = Path Length (cm)
Introduction
Self-assembly serves as a powerful tool for nanoparticle/composite synthesis and
recognition. The self-assembly process involves the construction of organized structures through
localized interactions among component parts [1]. The formation of these complex structures
gives rise to unique properties and applications by utilizing the properties of the individual
components involved in the self-assembly process. This process remains difficult to perform at
the nanoscale level. However, components such as DNA make nanostructure self-assembly
possible due to unique recognition, structural, and ease of manipulation characteristics. DNA-
guided self-assembly involves the self-assembly of specific nanoparticles, DNA and DNA
linkers to form complex structures that are dependent on the localized interactions and binding
processes between the three components [1]. The spatial arrangements of the resulting structures
may be controlled by this process due to DNA’s site-specific binding and structural properties, as
well as DNA’s ease of modification. By controlling the spatial arrangements, nanostructures with
specific geometries and patterns are made possible. Properties such as optical properties are
influenced by the size, shape, environment, and material composition of a structure; making
DNA-guided self-assembly a desired process for unique applications that expose these
properties. The assembly process begins with the attachment of a desired amount of DNA linkers
to desired nanoparticles and at specific angles to obtain a desired geometry. Colloidal gold (Au)
nanoparticles, with the DNA oligonucleotides attached strand-by-strand, may be given up to six-
fold symmetry according to a recent study [1]. Due to mutual exclusion and repulsion between
the DNA linkers, controlled angles of 90o and 180o were made possible; allowing the possible
formation of 3D shapes. These angles could be further modified by utilizing the physiochemical
interactions between DNA and the nanoparticles, such as steric hindrance and electrostatic
interactions between and within the components. In addition, modifying the length and twist of
the DNA helix may also be used to further control these angles and obtain a desired geometry.

9. 9 | P a g e
9
The potential degree of high accuracy and modification involved in this process ultimately
allows for the self-assembling formation of nanostructures with desired functions and arbitrary,
anisotropic shapes; including 3D shapes [1]. While this experiment doesn’t perform the DNA-
guided self-assembly technique, it aims to highlight the importance of its results. By analyzing
the impact of size and shape at the nanometer level, this experiment illustrates the degree at
which the properties of DNA-guided, self-assembling nanostructures are impacted by their size
and shape. This analysis ultimately provides an understanding of the variety of applications made
available by such a process having the ability to accurately form complex nanostructures of
different sizes and shapes in three dimensions.
There are numerous properties that may be influenced by the size and shape of a material.
Among these includes a material’s optical properties, of which are largely impacted by the size
and shape of a material; especially at the nanometer scale. Nanoparticles are typically smaller
than wavelengths of visible light and therefore are optically influenced by a significant degree
with even the slightest change in size or shape. These particles may both absorb and scatter light,
giving rise to extinction peaks (the sum of absorbance and scattering). The absorbance of
nanoparticles may be measured by a spectrophotometer. A spectrophotometer measures
absorbance values for a given wavelength range by sending a beam of light, composed of
photons, through a sample. When a photon reaches the sample, it may become absorbed by the
sample at a specific wavelength; reducing the number of photons in the beam of light that returns
to the spectrophotometer for analysis [3]. The spectrophotometer is therefore able to output
values of absorbance for each wavelength measured. By measuring the absorbance, or degree of
which a substance absorbs light at a specific wavelength, various nanoparticle characteristics
may also be determined. This feature is illustrated by the Beer-Lambert Law:
Equation(1): A = εCL
This equation indicates a linear relationship between the absorbance (A) and concentration (C) of
an absorbing species, as well as the path length (L) of the cuvette and the extinction coefficient
(ε) for a given species. By measuring some of these values, it is possible to calculate the
remaining values. For instance, the extinction coefficient (ε) of a species may be calculated by
calculating the slope of the absorbance readings for given concentrations. Due to the drastic
changes in these values at the nanometer level, it is also possible to determine attributes such as
size and purity by comparison techniques. This experiment performs dilutions in order to provide
comparisons and obtain these characteristics, while also referencing to the expected absorbance
values of the individual components. The dilutions cause a decrease in nanoparticle
concentration and therefore a decrease in peak absorbance values. Gold (Au) nanoparticles have
normal extinction peaks around 520-550 nm. These smaller particles mostly absorb light with
little scattering. More scattering occurs as the sizes of these particles increase, resulting in
broader extinction peaks at longer wavelengths due to increased optical cross-sections and
increased ratio of scattering to total extinction [2]. This phenomenon is called a red-shift, or a
shift in peak absorbance wavelengths towards the near-infrared range (650-900 nm). Thus, these
nanoparticles appear darker as their size increases. Comparison data may therefore be used to
determine the estimated size of these gold (Au) nanoparticles. The effects of light scattering may
also be seen by the changes in peak absorbance and absorbance wavelength range as a function
of gold nanoparticle size. Purity may also be determined using spectrophotometry for substances

10. 10 | P a g e
10
such as DNA and RNA by using ratios of absorbance values at different wavelengths. In the case
of DNA and RNA, the ratio of absorbance values at 260 nm and 280 nm is used to determine
purity, with a ratio of ~1.8 considered pure for DNA and ~2.0 for RNA [4]. For this experiment,
Gold (Au) nanoparticles with diameters of 5, 10, 30, and 60 nm are analyzed using dilutions and
spectrophotometry, with the diameter values concrete but unknown for each sample. The
extinction coefficients (ε) from the Beer-Lambert law are determined after absorbance readings
using the slopes of concentration vs. absorbance at peak wavelengths for each sample. The
diameters of the samples are determined by order of increasing diameter based on the absorbance
measurements and color changes. Finally, absorbance values for a DNA sample are given and
used to determine the purity of the sample.
Procedure
Equipment
 Ultra-Violet (UV)-Visible (Vis) Spectrophotometer
 Micropipettes- P20, 200, and 1000
 Micropipette tips
 1.5 mL micro centrifuge tubes
 Cuvettes- Quarts or equivalent
Reagents
 Citrate-Gold Nanoparticles (C-AuNPs; BBI Solutions):
-Diameters: 5, 10, 30, and 60 nm
-Concentration: 2.92 x 10-4 M
 Nanopure H2O (dd H2O; 18.2 MΩ*cm)
Procedure
1. Record the solution color before dilutions.
2. Dilute the solution by a factor of 2 by adding 100 µL of ddH2O to a new centrifuge tube,
then adding 100 µL of C-AuNPs from the previous centrifuge tube.
3. Dilute the solution by a factor of 2 by adding 100 µL of ddH2O to a new centrifuge tube,
then adding 100 µL of the sample in Step 2 and putting it in a new centrifuge tube.
4. Dilute the solution by a factor of 2 by adding 100 µL of ddH2O to a new centrifuge tube,
then adding 100 µL of the sample in Step 3 and putting it in a new centrifuge tube.
5. Dilute the solution by a factor of 2 by adding 100 µL of ddH2O to a new centrifuge tube,
then adding 100 µL of the sample in Step 4 and putting it in a new centrifuge tube.
6. Dilute the solution by a factor of 2 by adding 100 µL of ddH2O to a new centrifuge tube,
then adding 100 µL of the sample in Step 5 and putting it in a new centrifuge tube.
7. Add 90 µL of each sample (dilution) from the previous steps into a micro-cuvette, then
scan absorbance (A) using the UV-Vis Spectrophotometer.
Note: Each dilution factor was performed in triplicate to give three samples for each
dilution factor in order to eliminate error.

11. 11 | P a g e
11
Results
Dilution Factor Concentration (M) Peak Absorbance
(Wavelength)
0 2.92*10-4
1 1.46*10-4 Sample 1: 0.6105 (528 nm)
Sample 2: 0.6065 (526 nm)
Sample 3: 0.6124 (524 nm)
2 7.3*10-5 Sample 1: 0.2986 (527 nm)
Sample 2: 0.2994 (527 nm)
Sample 3: 0.3137 (527 nm)
3 3.65*10-5 Sample 1: 0.1362 (523 nm)
Sample 2: 0.1326 (525 nm)
Sample 3: 0.1467 (526 nm)
4 1.825*10-5 Sample 1: 0.0258 (529 nm)
Sample 2: 0.0599 (529 nm)
Sample 3: 0.0729 (527 nm)
5 9.125*10-6 Sample 1: 0.0122 (533 nm)
Sample 2: 0.0406 (523 nm)
Sample 3: 0.035 (530 nm)
Table (1): Dilution factor, concentration, and peak absorbance values for Group B.
Figure (1): Peak absorbance vs. concentration with linear trend lines for Group B.

12. 12 | P a g e
12
Figure (2): Average absorbance of the 3 samples vs. wavelength for Group B
Figure (3): Average absorbance for the 3 samples vs. wavelength for the first dilution for each
group.

13. 13 | P a g e
13
Figure (4): Absorbance vs. wavelength for the DNA sample.
Figure (5): Solution colors for each group in alphabetical order from left to right.
Discussion
The results of this experiment successfully illustrate the impact that size has on a
nanoparticles optical properties. The Beer-Lambert law allows the quantification and calculation
based on such properties. According to this law, absorbance and concentration are linearly
proportional. This relationship can be seen in table (1) and figure (2). Table (1) indicates
absorbance values approximately halving as the concentrations of the C-Au nanoparticles are
halved with each dilution. This same principle can be seen graphically in figure (2) to support the
application of the Beer-Lambert law for these samples. This law was therefore used to calculate
the extinction coefficient (ε) of each sample. By plotting peak absorbance versus concentration
in figure (1), we were able to find these coefficients as they is given by the slopes of these lines;
where A=εCL and the path length (L) was 1 cm. The coefficients are given as the slope of each
trend line in this figure. Sample 1 had an extinction coefficient of 0.2227 (M-1 cm-1), sample 2
had an extinction coefficient of 0.2109 (M-1 cm-1), and sample 3 had an extinction coefficient of

14. 14 | P a g e
14
0.2119 (M-1 cm-1). The average of these coefficients ((0.2227+0.2109+0.2119)/3) was 0.2152
(M-1 cm-1). The similarity in these values suggests a somewhat insignificant degree of error in the
experiment. Also, the R2 values of the trend lines used to calculate these values in figure (1) are
approximately equal to 1 to support little error in their calculations as well. Therefore, the
calculated extinction coefficients sufficiently estimate the true coefficients for these
nanoparticles. In total, this experiment was performed by four different groups with varying C-
AuNP diameters of either 5, 10, 30, or 60 nm for each group. These nanoparticles are expected to
give an increase in peak wavelength, towards the near-infared range, as the size of these particles
increases. The average absorbance values versus wavelength between the 3 samples and after the
first dilution for each group are given by figure (3). Group D had the smallest peak wavelength
around 520 nm. Therefore, this group likely measured the absorbance values for a solution of C-
AuNP’s with 5 nm diameters. Group B had the next smallest peak wavelength around 530 nm,
suggesting the use of solution composed of these nanoparticles with 10 nm diameters. There was
a large shift in peak wavelengths between groups B and D and groups A and C. This is supported
by the large size shift from 10 nm to 30 nm diameters. The gold nanoparticles of 30 nm diameter
were likely on the other end of this shift and measured by group A with a peak wavelength
around 620 nm. Therefore, group C can be suggested to have measured the nanoparticles with 60
nm diameters due to a peak wavelength around 630 nm. The colors of these solutions,
represented by figure (5), also support these claims. As previously stated, larger sizes indicate a
darker color for these nanoparticles. The far right solution, group D’s solution, appears the most
transparent while the solution second from the right, group C’s solution, appeared the darkest.
The absorbance versus wavelength measurements for our DNA sample are given in figure (4).
These measurements were used to determine the purity of this sample by calculating the ratio of
absorbance values at 260 nm and 280 nm. The measured absorbance values were 0.513 at 260
nm and 0.302 at 280 nm with the ratio (0.513/0.302) around 1.7. With a ratio of 1.8 indicating a
pure DNA sample, the measured ratio therefore suggests a quite pure DNA sample.
References
[1] Kim, Jin-Woo. Self-Assembly of Nanocomposites. Nanotechnology Laboratory. University
of Arkansas, Aug. 2015. Web. 14 Oct. 2015.
[2] "Gold Nanoparticles: Optical Properties." NanoComposix. N.p., n.d. Web. 14 Oct. 2015.
[3] Blauch, David M. "Spectrophotometry." Spectrophotometry Version 2.1. Davidson
University, 2000. Web. 14 Oct. 2015.
[4] "260/280 and 260/230 Ratios NanoDrop® ND-1000 and ND-8000 8-Sample
Spectrophotometers." Technical Support Bulletin (2007): n. pag. NanoDrop. Web. 14 Oct. 2015.

15. 15 | P a g e
15
Lab 8: Protein Concentration
Jacob Feste
010617389
3/29/15
Objective
The objective of this experiment was to both determine and adjust the concentration of a
purified protein using centrifugal filtration units. The final adjustment goal of this experiment is
to reach a protein concentration of 1mg/ml by using concentrators and repeating
concentration, re-suspension, and protein analysis. To adjust the protein concentration, the
concentrator PBS was applied to the protein and the volume of the added PBS was adjusted in
order to reach the desired concentration. This mixture then underwent centrifugal filtration, of
which separates the mixture by size to separate the protein and solvent. Two-fold serial dilution
was then performed for comparison purposes for both the purified protein and a standard
protein solution with a known initial protein concentration of 2 mg/ml. This method reduced
the protein concentrations in half with each dilution. To determine the concentration of the
purified protein, BCA assay was used. This method applies bicinchoninic acid (BCA) and cupric
ions to the protein, of which the cupric ions become reduced by the protein’s peptide bonds
and the BCA reacts with these reduced cupric ions. The absorbance of the mixture is used to
give concentrations. These steps were repeated until the desired 1mg/ml concentration was
reached. The expected outcome of this experiment, reaching a purified protein concentration
of 1 mg/ml, is successful when the BCA assay absorbance readings at 562nm display very similar
readings for the non-diluted purified protein sample and the first dilution of the standard
protein solution. The standard protein solution has an initial protein concentration of 2 mg/ml
and therefore a protein concentration of 1 mg/ml after its first dilution to give accurate
absorbance readings of what the purified protein should read to be considered at 1 mg/ml
protein concentration.
Materials
1. PBS
2. Centrifugal Filtration Unit
3. 6.25ml Protein Solution
4. Micropipettes
5. Sample Reservoir
6. 10ml DI Water
7. 96 Well Plate
8. 200µl Standard Protein Solution (2 mg/ml)
9. 1400µl Water or Buffer

16. 16 | P a g e
16
10. 5ml Reagent A
11. 100µl Reagent B
12. Incubator
13. Absorbance Detector
Procedures
Concentrating proteins:
1. Wash the filtration unit by adding 10ml of PBS to the tube and centrifugation at 4000
RPM and 4ºC for 5 min
2. Discard the buffer from the centrifuge tube and the sample reservoir
3. Add 6.25ml of protein solution (0.8mg/ml) to the sample reservoir
4. Increase the sample volume to 10ml by adding appropriate amount of PBS to sample
solution
5. Run the tube through centrifugation at 4000 RPM and 4ºC for 15 min
6. Discard the waste solution from the waste reservoir
7. Now add 10ml of PBS to the sample reservoir
8. Repeat step 5
9. Discard the waste solution from the waste reservoir
10. Collect the sample (should be around 4 to 5 ml) from sample reservoir
11. Collected sample concentration can be determined using BCA assay
12. Once you obtain the concentration adjust the volume using PBS to get the required
concentration
Regenerating the filters:
1. Add 10ml PBS to the sample reservoir
2. Centrifuge at 4000 RPM for 5 min
3. Discard PBS from both waste and sample reservoir
4. Repeat steps 1-3
5. Now add 10ml DI water to sample reservoir
6. Centrifuge at 4000 RPM for 5 min
7. Discard water from both waste and sample reservoir 8. Close the filter unit and return to
TA
Prepare Standards:
1. Use one 96 well plate for BCA assay.
2. Add 100 μl of standard protein solution to row A of columns 1 and 2
3. Add 100 μl of your protein solution to row A of columns 3 and 4
4. Add 50 μl of water or buffer to the rows B-H of columns 1-4
5. Perform two-fold serial dilutions:
o Remove 50 μl from the row A, add to row B

17. 17 | P a g e
17
o Mix by pipetting 3x
o Repeat through row G
o Leave the last row with buffer only
BCA Assay:
1. Prepare BCA working reagent by adding 5ml of reagent A to 100ul of reagent B
2. Transfer 25 μl of standard protein solution from A1 and A2 to A5 and A6 in the 96 well-
plate. Repeat this for the other rows (B,C,D,E,F,G,H) for standard solutions.
3. Transfer 25 μl of your protein solution from A3 and A4 to A7 and A8 in the 96 well-plate.
Repeat this for the other rows (B,C,D,E,F,G,H) for your protein solutions.
4. Add 200 μl of working reagents to all wells
5. Place the well plate in 37ºC incubator for 30 min until the solution turns purple
6. Now take absorbance reading at 562 nm
7. Compare sample readings to standard curve to calculate protein concentration
Results
Standard
Protein
Solution
Sample
1
Standard
Protein
Solution
Sample
2
Purified
Protein
Solution
Sample
1
Purified
Protein
Solution
Sample
2
1.89 1.747 1.014 1.097 Read 1:562
1.897 1.751 1.015 1.1 Read 2:562
1.044 1.001 0.51 0.456 Read 1:562
1.046 1.001 0.511 0.457 Read 2:562
0.534 0.609 0.312 0.363 Read 1:562
0.536 0.612 0.312 0.363 Read 2:562
0.315 0.359 0.197 0.239 Read 1:562
0.316 0.357 0.196 0.239 Read 2:562
0.215 0.219 0.157 0.182 Read 1:562
0.214 0.218 0.157 0.181 Read 2:562
0.19 0.176 0.154 0.149 Read 1:562
0.188 0.173 0.152 0.148 Read 2:562
0.155 0.159 0.145 0.167 Read 1:562
0.149 0.153 0.138 0.161 Read 2:562
0.155 0.152 0.145 0.151 Read 1:562
0.143 0.141 0.135 0.14 Read 2:562
Figure 1: BCA assay absorbance readings for both the known standard protein solution samples
and the unknown purified protein solution samples.

18. 18 | P a g e
18
Discussion and Conclusion
The objective of this experiment was to obtain a purified protein concentration of 1 mg/ml. In
order to accomplish this objective, adjustments of the original protein solution at 0.8 mg/ml
were made. This was done by adding volumes of PBS to the solution and then using centrifugal
filtration to separate the protein and solution. PBS was used due to its ability to dissolve a
protein and store it in its solution. Therefore, by adding PBS to the protein solution the proteins
dissolve and store themselves in their solution. The dissolved proteins are then able to be
separated by centrifugal filtration, separating some of the solvent from its proteins. This
process of adding PBS was repeated until enough solvent was separated from the proteins to
increase the concentration to 1 mg/ml. After analyzing the BCA assay absorbance readings in
Figure 1, it can be concluded that this objective was met. As stated previously, a protein
concentration of 1 mg/ml is indicated by absorbance readings similar to those of the first two-
fold dilution of the known standard protein solution due to the original 2 mg/ml concentration
being cut in half to 1 mg/ml after dilution. Figure 1 illustrates the absorbance readings of the
standard protein solution samples after their first dilution to be between 1.001 and 1.046. It
also illustrates the absorbance readings of the non-diluted purified protein samples to be
between 1.014 and 1.1. While there was certainly a small degree of error, these results are
similar enough to conclude that the purified protein solution samples were around 1 mg/ml
due to their similar absorbance readings with the known 1 mg/ml readings. This assumption is
further supported in Figure 1 by the pattern of similar absorbance readings for the known
standard protein solution and those of one less dilution of the purified protein solution. While
these readings were close enough to make this assumption, there was also a small degree of
inaccuracy. This inaccuracy is indicated by the degree of small difference in absorbance
readings for the samples of the same type, as well as the small difference in absorbance
readings for the correlating wells of the different types. Reasons for the difference in readings
for the samples of the same type could likely be due to inaccuracy of the absorbance detector.
It could also be due to the small difference in length the 562nm light has to travel for each well
due to their very small differences from each other. The difference could also be due to
inaccurate measurements as well as small contamination from molecules such as dust particles
due to the solutions being exposed to the air environment. Reasons for the difference in
readings for the previously explained correlating wells of the different sample types could be
similar to those listed above. The biggest degree of difference, however, was most likely due to
error in protein concentration adjustments for the purified protein solution. It is very difficult to
adjust the protein concentration to exactly 1 mg/ml, therefore most of the inaccuracy is likely
due to the purified protein solution’s protein concentration differing slightly from 1 mg/ml. In
conclusion, however, the experiment was performed successfully and the objectives were met
despite this slight inaccuracy. The purified protein solution had its protein concentration
successfully adjusted from 0.8 mg/ml to around 1 mg/ml, supported by the BCA absorbance
results.

19. 19 | P a g e
19
Lab 7: Protein Qualification
Jacob Feste
010617389
3/15/15
Objective
The objective of this experiment was to determine the protein concentration of an unknown
protein sample, as well as its protein’s absorption coefficient. This objective was obtained by
performing a serial dilution of both known protein concentration standard samples and the
unknown protein concentration protein samples, and then by performing the protein
quantification methods of UV absorbance and the Bicinchoninic Acid (BCA) method. The results
of both sample types from these methods may then be compared statistically in order to
estimate the protein concentration of the unknown protein sample, as well as the absorption
coefficient. The standard solution originally contained a protein concentration of 1mg/ml, while
serial dilution was performed to cut the concentration in half each time for both sample types
until a concentration of 1/64 was obtained, along with a blank for reference. This serial dilution
method allows for the trend comparison of both samples types in the two quantification
methods performed in order to determine the concentration of the unknown sample type. The
BCA quantification method utilizes the ability of the peptide bonds, found in proteins, to reduce
cupric ions, of which BCA reacts with and absorbs at 560nm. Therefore this method displays the
overall protein content by the amount of BCA and cupric ion reactions that took place, which is
indicated as increasing with increasing absorbance. Also, the UV absorbance method was
performed on the known protein concentration, standard samples in order to determine their
protein and DNA/RNA content after dilution for comparison. Both 280nm and 260nm
wavelength tests were performed as most proteins absorb maximally at 280nm while DNA/RNA
absorb maximally at 260nm wavelengths, and therefore allows for the comparison of each
amount as well as an overall reference for the unknown concentration sample type. Therefore,
the UV absorbance results should decrease with decreasing concentrations for both 280nm and
260nm as less and less amounts of protein and DNA/RNA are present for each dilution. The
same pattern should be observed in the 560nm BCA test as well for the same reason, while the
trends from each test should allow for the correct concentration identity of the unknown
protein sample type.

20. 20 | P a g e
20
Materials
1. BCA reagents A and B
2. Two clear 96 well plates
3. Biotek Take 3 Plate
4. Bovine serum albumin standard solution
5. Unknown protein solution
Procedure
Prepare Reagents:
Add 10 ml of reagent A and 200 μl of Reagent B to a 15 ml tube to create the working reagent
(WR)
Prepare Standards:
1. Label two 96 well plates as Plate 1 and Plate 2
2. Add 100 μl of standard solution to row A of columns 1 and 2 of Plate 1
3. Add 100 μl of your protein solution to row A of columns 3 and 4 of Plate 1
4. Add 50 μl of buffer to the rows B-I of columns 1-4
5. Perform two-fold serial dilutions
a. Remove 50 μl from the row A, add to row B
b. Mix by pipetting 3x
c. Repeat through row H
d. Leave the last row with buffer only
BCA Assay:
1. Transfer 25 μl from all wells of Plate 1 to their corresponding wells in Plate 2
a. Set Plate 1 aside for use in the UV absorbance assay
2. Add 175 μl of WR to all wells of columns 1-4 of Plate 1
3. Place on shaker for 30 minutes at 37 C
4. Record absorbance at 562 nm
UV Absorbance:
1. Transfer 2 μl of your standards in columns 1-2 to the Take3 plate
2. Use the bottom row as your blank
3. Record absorbance at 280 and 260 nm after path length and blank correction
4. Repeat with the samples in columns 3-4

21. 21 | P a g e
21
Results
Figure 1: BCA protein concentration vs. absorbance results at 560nm for the known standard
and unknown protein solutions.
Figure 2: UV protein concentration vs. absorbance results at 260nm for the known standard
solution.
Protein Solution 1:
y = 1.0561e-0.308x
R² = 0.822
Protein Solution 2:
y = 0.6691e-0.123x
R² = 0.2687
0
0.5
1
1.5
2
2.5
1 0.5 0.25 0.125 0.0625 0.03125 0.015625 0
Absorbance
Protein Concentration (mg/ml)
BCA Absorbancevs. Concentration at560nm
Standard Solution 1 Standard Solution 2
Protein Solution 1 Protein Solution 2
Expon. (Protein Solution 1) Expon. (Protein Solution 2)
0
0.1
0.2
0.3
0.4
0.5
0.6
1 0.5 0.25 0.125 0.0625 0.03125 0.015625 0
Absorbance
Protein Concentration (mg/ml)
UV Absorbancevs. Protein Concentration at260nm
Standard Solution 1 Standard Solution 2

22. 22 | P a g e
22
Figure 3: UV protein concentration vs. absorbance results at 280nm for the known standard
solution.
Conclusion
The objective of this experiment was to determine the protein quantification, specifically
concentration, of an unknown protein solution. By analyzing the results of this experiment, this
objective was primarily met. The BCA results, illustrated by Figure 1, gives a nice estimate of the
protein concentration of our unknown sample. Although the second unknown protein solution
sample gave results of error, indicated by its R^2 value from its standard curve, the first
unknown protein solution sample gave accurate results. The results of its standard curve gave
an initial concentration of about 0.5 mg/ml of protein, or half of the standard solution.
Therefore, after the first dilution of the standard sample of which cut its protein concentration
in half, the rest of the graph should look similar for each solution as it does in Figure 1. This also
indicates that our dilutions did an excellent job at predicting the starting concentration of our
unknown protein solutions, as the dilution factor almost exactly parallels the protein
concentrations in Figure 1 and therefore an extra plot was not necessary as it is almost
identical. The UV absorbance results, illustrated by Figure 2 and Figure 3, were of much error
and therefore cannot be accurately used for analysis. These results should give a decrease in
absorbance with decreasing protein or DNA/RNA concentration, while they give an inaccurate
wave of data. Therefore, the absorption coefficient is calculated to be around 0.06-0.08
ml/(mg*cm) for the first data point, but quickly increases with each point instead of remaining
the same due to similar absorbance values for each decreasing concentration. Unfortunately,
this makes the calculated absorbance coefficient values irrelevant and also makes any other
accurate references from these figures impossible. This huge source of error was likely due to
improperly “zeroing” before the UV absorbance, as our blank used to zero it was accidentally
not a correct blank to use. This was due to error while diluting in which the blank also included
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Absorbance
Protein Concentration (mg/ml)
UV Absorbancevs. Protein Concentration at
280nm
Standard Solution 1
Standard Solution 2

23. 23 | P a g e
23
the last dilution of the sample, and therefore was not truly a blank which through off every
possible measurement. Other errors in the UV absorbance tests were also likely related to
diluting. Errors in the BCA test were much less apparent as it gave accurate protein
concentration results. However, errors in diluting could have also occurred as well as other
procedures in the actual BCA assay process. The differing values between the two samples for
each sample type indicate that a small amount of error was present and likely due to dilution,
but overall it was not significant enough to conclude it inaccurate. While the absorption
coefficient could not be measured due to UV absorption error, other tests such as a
fluorescence detection test could have been used to detect this value. In conclusion, dilutions
with the BCA assay test gave us an accurate initial protein concentration of around 0.5 mg/ml
for our unknown sample. However the other quantification desirable, the tested protein’s
absorption coefficient, could not be accurately measured from the UV absorbance tests due to
error and therefore this objective was not met.