Austin Van Niel

Biochemistry Lab Section 10

Partner: Emma Mastrobattista
TA:Olijora Rezhdo
2/13/2017

Protein Concentration and SDS-PAGE

1. The absorbance spectra of tube 7 was sketched in the lab notebook and it
showed the expected values for a Lowry assay as shown on page 13 of the NanoDrop
handbook. Tube 7 contained 10uL of 0.50mg/mL bovine serum albumin, 50uL of alkaline
reagent, and 5uL of Folin reagent. There was a small section of negative values (less
than 5Au) before A450 as expected because blue light is being reflected and then it
curves upwards into positive values asymptotically. Compared to the expected curve it
does not level off around 600nm at 0.1Au and appears to continue to rise as the
concentration increases possibly due to contaminants in the tube. Of the contaminants
listed in the NanoDrop handbook it does not follow any of the given contaminated
spectra but this could have been due to a non-homogeneous solution which was
indicated in the NanoDrop handbook to cause significant deviation in the expected
spectra. The sample was a blue color because the proteins reduced the Folin reagent.
The wavelength used for additional calculations was 650nm as per the standard Lowry
assay, the spectra were normalized to 405nm per the Nanodrop handbook. These
values were appropriate because it was visualized that the absorbance at 405nm was
very low and the absorbance at 650nm was very high, comparatively. This was expected
because the absorbance of copper is 650nm which indicates that the tetradentate
copper-protein complex was formed from the reaction with cupric sulfate resulting in the
blue color. The other wavelengths that the software could have used to calculate the
protein concentrations could have been anywhere above 405nm which was detectable
above the background but realistically this should be capped at 750 nm as there was no
absorbance shown above this value.

2.
 Tube # mg/mL A650

1 0.000 0.02

2 0.020 0.04

3 0.050 0.09

4 0.10 0.11

5 0.15 0.12

6 0.20 0.18

7 0.50 0.39

8 Out of range 1.58

9 Out of range 0.39

10 0.059 0.07

11 Out of range 0.53

12 Out of range 0.00

This data was gathered from a different lab group because our group had trouble with
blanking the Nanodrop and there were issues with the data being out of range. In order to save
time the data was taken from the group of Sara Chung and Ken Kim and was used in all later
calculations. The data labeled Out of range is because the concentration of protein was too
high or too low for the set values.

3.
For tubes 9 and 10 the absorbance values at 650nm of 0.39Au and 0.07Au respectively fall
within the values specified by the standard curve, tube 8 was out of range for the values in the
standard curve with an absorbance of 1.58Au. From the standard curve it can be estimated that
the concentration for tube 10 to be 0.0542mg/mL using the best fit equation with the absorbance
value of 0.07 which was confirmed by the graph. Since tube 10 is 100 times diluted from tube 8,
meaning that the concentration is 100 times greater than tube 10, giving a value of 5.42mg/mL.
Tube 9 had an absorbance of 0.39 and from the best fit equation it can estimated to have a
concentration of approximately 0.500mg/mL which would give an estimation of tube 8 to be
5.00mg/mL since it was a 10 times dilution. It can be posited that the actual concentration of
tube 8 is between 5.00 and 5.42 mg/mL given the information gathered from the standard curve.

4.
One recommendation for future experimentation was that there was not enough time
allotted to allow the mixtures to incubate and react to form the necessary compounds for
spectral analysis. In the future it may be possible to incubate for a shorter time while agitating
the mixture to speed up the reaction. The solutions could also have been incubated at a higher
temperature to increase the reaction velocity without denaturing the proteins. The room
temperature incubation was to protect against denaturing the proteins and the Folin reagent
from denaturing but an increase from room temperature to 40 could have been done without
risking the efficacy of the assay.

The reliability of the standard curve may have been increased by performing multiple
readings on the tubes. This would have improved the linear correlation constant to have more
data points to counteract any outliers in the data and may have pointed out if there were any
issues with contaminants of homogeneity more readily so that they could have been dealt with
before completing any estimations. This way several estimations of the unknowns could have
been made and the average taken in order to increase the accuracy of them and reduce
random error.
If students had prepared all of the protein dilutions for the standard curve it may have
improved the learning experience by allowing us to use the equations for serial dilutions that
have been taught in class but never used in a practical setting. This would have also given us
the possibility to catch any mistakes in pipetting should the spectra be far off from what we
expected in the dilutions of known concentration. This would however introduce more sources of
error and have taken more time which was an issue especially with the long incubation times
that the assay requires as written in the lab manual.

5.

This is an SDS-PAGE gel of various proteins used in previous labs. The gel is 7.5%
polyacrylamide and was run for 40 minutes at 150 volts. The sample loading buffer contained
sodium dodecyl sulfate to denature any non-covalent interactions found in the samples and to
 coat the proteins in negative charge. It also contained beta-mercaptoethanol which will break
the disulfide bridges found in the proteins by reducing them. The loading buffer also contains a
dye used to monitor the migration of the sample. Lanes 1 and 8 contained 10uL of Bio-Rad
Precision Plus Protein Unstained Standards molecular weight marker. 15uL of the provided
standards were mixed with 15uL of loading dye, then 15uL of this mixture was loaded into the
remaining wells, so 7.5uL of the protein standard is present within the wells. Lanes 2 and 9
contained 7.5uL of bovine serum albumin, lane 9 is a duplicate of lane 2 because there was a
question of error in loading the sample into lane 2. Lane 3 contained 7.5uL of alkaline
phosphatase. Lane 4 contained 7.5uL of chymotrypsin. Lane 5 contained 7.5uL of malate
dehydrogenase. Lane 6 contained 7.5uL of strawberry DNA isolated by the precipitation method.
Finally, lane 7 contained 7.5uL of strawberry DNA isolated by the column method (a sample
from another group was used).

6.
The sample of bovine serum albumin (BSA) (wells 2 and 9) migrated as expected since
it was the heaviest at 66.5kDa and migrated the least. The molecular weight marker indicated
that the BSA should be slightly lower than the 4th band which indicates 75kDa which is where
the BSA band migrated. The sample of alkaline phosphatase did not migrate as something with
a chain size of 53kDa which would typically migrate to just above the 5th band of the marker
indicating 50kDa. In the gel it moved past the 5th band in a smear which makes it unclear as to
where the band ended which could indicate that there was other proteins or molecules in the
sample. The sample of chymotrypsin in lane 4 has three chains that become separated when
they are denatured by beta-mercaptoethanol. The smallest chain at 1.2kDa migrated the
farthest and was shown by a band but there were no bands shown for the other chains of 13.8
and 10.1kDa are either still unseparated from the small chain which would indicate that the gel
should have been run for a longer period of time to increase the spatial contrast or that it should
have been left to react with the loading buffer for a longer period of time. The sample of malate
dehydrogenase in lane 5 did not migrate as expected as its chain size of 33kDa should put the
band between 6th and 7th bands it instead migrated all the way down with a smear between
bands 7 and 8. The DNA isolated from lab 1A did not show any bands on the gel which indicates
that there are no protein contaminants that are larger than 10kDa and that the concentration of
DNA in these samples is so low that it is not immediately visible on cursory inspection.There
were no significant sources of error or contamination that invalidate the results of the SDS-
PAGE gel other than some of the samples not containing chains of the indicated length.

7.

Sample A280 (Au) A260/A280 Concentration Volume (uL) Mass (ug)

(Au) (ug/uL)
Bovine Serum 0.500 0.88 499 7.5 3742.5
Albumin (BSA)

Alkaline 3.00 0.73 2998 7.5 22485

phosphatase

Chymotrypsin 2.32 0.68 2317 7.5 17377.5

Malate 1.50 1.28 1500 7.5 11250

dehydrogenase

Strawberry DNA 5.55 1.27 5554 7.5 41655

(precipitation)

Strawberry DNA 0.18 1.41 180 7.5 1350

(column)
The greater the mass of the protein the brighter the band in the gel will appear after
staining because this correlates to a higher concentration of protein which is proportional to the
amount of visible dye. From the data and the gel that was taken it would appear however that
the mass and the brightness of the band is uncorrelated. In lane 4 which contained
chymotrypsin it was calculated to have a mass of 17377.5 ug meaning that it should be much
brighter than lane 2 which contained BSA which was calculated to have a mass of 3742.5ug, but
it does not appear brighter. If anything it appears with the same brightness qualitatively. This is
also apparent in lane 6 which had a mass of 41655 ug, the largest mass found, which should be
the brightest band on the whole gel, but it is barely present at all and the band seems to be
residual loading dye.
From the data that was obtained by this sequence of experiments, it is difficult to tell
examine the proclivity for allowing contaminants into the DNA samples because we received
little accurate information from the gels or the spectral images to indicate that there was more
protein versus contaminants or the types of contaminants. The SDS-PAGE did not show any
bands that would indicate that there was DNA in either sample, but this also indicated that if
there were any contaminants they were small enough not to get caught in the gel in place of the
desired protein. The Lowry assay that was performed showed out of range values for the
strawberry DNA sample concentrations for both precipitation and column methods and very little
absorbance. This was probably because the contents of these samples did not have an
absorbance at the expected wavelength of nucleic acids at 650 nm. The A280 assay showed
that the strawberry samples did not show the expected spectra at the absorbance of 280nm.
There could be possible contaminants for the A280 spectra including carbohydrates and
peptides because it was a general sample taken from the strawberries which would have
created the values found had either method not been performed with precision. Guanine
residues are commonly used in the column method for DNA isolation which causes a slight peak
at 230 nm which we found, but very small in magnetude. Contaminants for the other methods
are impossible to speculate because the data were out of range and the spectra were so far off
from the expected graphs that it could be any of these, all of these, or some combination of
these. There is no information that indicates whether it is out of range above or below the set
spectral parameters.