Biol3140 Lab 2
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Tabashir ChowdhuryID: 211219136
BIOL3140
Advanced Biochemistry and Molecular Genetics Laboratory
Laboratory Report 2
Protein Analysis for alpha Amylase
TA: Mez
Date of Submission: 17/10/11
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Tabashir ChowdhuryID: 211219136
Abstract
A number of samples of proteins including bacterial cell lysates proteins and
commercially available alpha amylase proteins from 3 different sources were separated
using SDS-PAGE to determine relative molecular weight of the proteins, followed by
western blotting using primary antibodies for alpha amylase to detect enzyme activity in
the samples. Polyclonal antihuman alpha amylase antibodies and anti Bacillus
amyloliquefaciens antibodies were used. Unknown sample 1, alpha amylase samples
from A. oryzae and porcine pancreas as well as the B. amyloliquefaciens crude extract
produced bands in western blot, indicating the presence of alpha amylase activity in these
samples. Presence of band for alpha amylase from porcine pancreas shows regions of
conserved amino acid sequences shared between the mammalian amylase proteins. The
lack of the band for Bacillus licheniformis amylase indicates that there is little or no
amino acid sequence homology between the two bacterial amylase proteins. The amylase
in the bacterial cell lysate was also identified conclusively from the other proteins by
virtue of the anti alpha amylase antibody interaction with the amylase.
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Tabashir ChowdhuryID: 211219136
Introduction
The objective of this experiment was to analyze protein compositions of several -
amylase containing samples including cell lysates of Bacillus amyloliquefaciens and
Bacillus licheniformis, a number of commercially available alpha amylase samples1 and
saliva samples. Amylases are important enzymes obtained form various sources, such as
plants, animals, fungi and bacteria2. The main function of α-amylases is to catalyze the
breakdown of amylose and amylopectin through the hydrolysis of internal alpha-1, 4-
glycosidic linkages3, 4.
The proteins in the samples were separated according to size using SDS-PAGE and their
molecular sizes were determined by constructing a standard curve that plots relative
mobility against the molecular weight of the proteins in the pre-stained protein marker.
Relative mobility refers to the movement of a type of polypeptide through a gel relative
to other protein bands in the gel. It is the distance migrated by a band divided by the
distance migrated by the dye front. Using the standard curve the molecular sizes of the
proteins (-amylases) from each sample can be estimated.
SDS-PAGE also referred to, as denaturing gel electrophoresis1 is ideal for analysis of
proteins as it uses a detergent SDS and a reducing agent ß-mercaptoethanol to break
down the native structure of the proteins and causing all proteins to have the same shape 1.
Performing a Polyacrylamide gel electrophoresis (PAGE) on the denatured proteins
causes the proteins to be separated solely based on their molecular size.
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Tabashir ChowdhuryID: 211219136
The second part of the experiment involved the detection of the separated proteins using
immunoblotting or a western blot. The proteins separated by SDS-PAGE were transferred
to a membrane, which was then incubated with antibodies specific to the proteins of
interest (in this case –amylase). The detection of the protein was carried out via a
secondary antibody (Anti-rabbit IgG) specific for the primary antibody (Anti -amylase).
The secondary antibody is attached to an enzyme (alkaline phosphatase) that converts a
soluble colorless substrate (BCIP) into an insoluble colored product1. As a result dark
blue bands appear where there is interaction between the proteins and antibodies.
Analysis of the western blot allows us to conclusively identify which of the bands in
samples of the Bacillus cells and the human saliva samples was an -amylase. This is
possible due to the strong and specific antigen-antibody interactions.
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Tabashir ChowdhuryID: 211219136
Materials and Methods
The experiment is carried out according to the protocol listed in the BIOL 3140
Laboratory manual, Biotechnology: DNA to Protein- A Laboratory Project in Molecular
Biology by Theresa Thiel, et al. 2002. The changes made to the protocol are mentioned
below:
Preparation of Samples (pages 50-51, BIOL 3140 Lab Manual)
- 100µl of TE (10mM Tris-HCl, pH 8, 1mM EDTA) is added to each of the 1.5ml
tubes containing the bacterial culture.
- 100µl of dry beads were used which were added to the bacterial cell crude extracts.
- 15µl of each bacillus lysate is transferred to a new tube followed by 15µl of 2X SDS-
PAGE loading buffer
- 15µl of saliva sample is transferred to a 1.5ml tube and 15µl of 2X SDS-PAGE
loading buffer is added to the sample.
- 15µl of each of the 3 commercial amylases and 2 unknown samples were added to 3
eppendorf tubes followed by 15µ of 2X SDS- PAGE loading buffer.
Staining the polyacrylamide gel (pages 52-53, BIOL 3140 Lab Manual)
- After the gel is submerged in the stain solution it is not microwaved.
- The gel is not microwaved after adding the destain solution.
Transfer proteins from gel to membrane (pages 56-58, BIOL 3140 Lab manual).
- PVDF membrane is used instead of nitrocellulose membrane and the membrane is
soaked in 20ml of methanol.
- The gel is covered with 15-20ml of western transfer buffer.
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Tabashir ChowdhuryID: 211219136
- 4 sheets of the absorbent paper are placed in a separate container along with the
sponges in 50-100ml of western transfer buffer.
- The transfer sandwich containing the membrane is allowed to transfer overnight at
40C with a current of 40mA.
Detect Proteins Immunologically (pages 58-59, BIOL 3140 Lab Manual).
- 20ml of the primary antibody solution is added to the membrane after the blocking
buffer is discarded.
- 25-30 ml of TBST is used on each occasion to rinse the membrane after discarding
the primary antibody solution.
- 20 ml of the secondary antibody solution is added to the membrane after all the wash
solutions are discarded. The membrane is incubated for an hour.
- The membrane is rinsed twice with 30 ml of TBST after the secondary antibody
solution is discarded.
- 30ml of AP reaction buffer is added to the membrane after rinsing with TBST.
- After the BCIP/NBT premixed solution is discarded the membrane is briefly rinsed
with dH2O. The dH2O is then discarded and the membrane is rinsed gently for 2-3
minutes, and then allowed to dry.
-
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Tabashir ChowdhuryID: 211219136
Results
After the proteins were separated using SDS-PAGE one of the polyacrylamide gels were
stained with Coomassie blue staining solution (Figure 1). The broad range protein marker
(NEB P7701) with proteins of known molecular weight in lane 1 was used to construct a
standard curve with relative mobility of proteins Rf on the x-axis vs. the molecular weight
in Kilo Daltons Kd on the y-axis. (Table 1) shows the distances migrated by the proteins
of known molecular weight in the protein marker and the relative mobility for each of the
proteins. The relative mobility was calculated by measuring the distance migrated by
each band with the distance migrated by the tracking dye (97mm).
After that the distance migrated by the bands for each of the sample proteins were
measured and used to calculate the relative mobility of the proteins. Lane 2 containing
the Unknown sample 2 showed no bands on the gel. Lane 3 was loaded with unknown
sample 1 showed a very thick band, which migrated 38 mm and had a relative mobility of
0.388. Lane 4 contained the Bacillus amyloliquefaciens crude extract that showed a band
at 36mm with a relative mobility of 0.367. Lane 5 was loaded with alpha amylase from
Aspergillus oryzae, showed two bands at 37mm and 42 mm respectively with relative
mobility of 0.378 and 0.429. Lane 6 contained the Bacillus licheniformis alpha amylase
with a very thick band that migrated 39 mm with a relative mobility of 0.402. Lane 7
contained a saliva sample that showed two bands. The first one migrated a distance of
only 5.5mm with a relative migration of 0.056, while the second band migrated a distance
of 39mm with a relative migration of 0.402. Lane 8 contained the alpha amylase from
porcine pancreas with a very faint band at 37 mm with a relative migration of 0.381. Lane
9 contained the crude extract from bacillus licheniformis, and showed several bands.
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Tabashir ChowdhuryID: 211219136
Figure 1 The Coomasie-stained –SDS-PAGE shows 2 protein markers of known size on each side of the gel. The broad range (NEB P7701) has been shown along with the known molecular weights of its bands. Each band is tagged with a letter (a-f. The two unknown samples of this experiment are in lane 2 and 3, shown as. Two different lysates of B. licheniformis were run in lanes 4 and 9 with B. amyloliquefacines crude sample in lane 4 and B. licheniformis crude extract in lane 9. Three commercially purified α-amylase samples were run in lane 5, 6 and 8. With α-amylase from porcine pancreas (Sigma A3176) in lane 8, α-amylase from Aspergillus oryzae (Sigma A6211) in lane 5 and α-amylase from B. licheniformis (Sigma A4551) in lane 6. A human saliva sample was run in lane7 and finally a second pre-stained protein marker (NEB P7711S) was placed in lane10. Horizontal yellow lines highlight the bands observed in each lane and the 3 bands observed in commercial α-amylases are further highlighted with “α” on each band
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The first one migrated only 8.5 mm with a relative mobility of 0.088, the second and
third bands migrated a distances of 40mm and 42 mm respectively, with relative mobility
of 0.412 and 0.433. Finally lane 10 was loaded with a prestained protein ladder.
Table 1 illustrates the absolute distances in mm for each of the bands in the Broad range
protein marker as well as their respective molecular weights and the relative mobility of
each band. The Standard curve (Figure 2) was constructed using data from table 1. A line
of best fit is obtained from the curve, which was used to determine the molecular weights
of the bands from all the samples. Table 3 shows the absolute distance migrated by each
of the bands from the samples as well their relative mobility and finally their molecular
weight, which was determined using the equation of the line of best fit of the standard
curve.
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Tabashir ChowdhuryID: 211219136
Table 1: Molecular Weight and Migration Distance of broad range protein marker (NEB P7701) along with the calculated relative migration distance and molecular weight. Relative distance was calculated through division of absolute distance travelled by the distance travelled by the tracking dye.
Protein Marker of known molecular
weight in Kd
Distance migrated in gel
(mm)Relative Mobility Rf
(a) Phosphorylase b (97.4 kd) 8 0.08
(b) Bovine serum albumin (66.2 kd) 18 0.1856
(c) ovalbumin (42.7 kd) 27 0.278
(d) carbonic anhydrase (31kd) 37 0.381
(e) trypsin inhibitor (21.5 kd) 55 0.567
(f) lysozyme (14.4 kd) 64 0.6598
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0 0.1 0.2 0.3 0.4 0.5 0.6 0.70
20
40
60
80
100
120
f(x) = − 130.715142311955 x + 92.4034261949899R² = 0.870163312282386
Relative Mobility Rf
Mol
ecu
lar
Wei
ght
(kd
)
Figure 2 This figure shows the standard Curve for molecular weight against relative migration distance of Broad range protein ladder (NEB P7701). Line of the best fit and its formula show a pattern migration of separated proteins based on their molecular weight.
Table 2 This table shows the samples with one or more band on the SDS-PAGE, along with the name of each sample, absolute migration distance of each sample, relative migration and each sample’s estimated molecular weight.
LaneNumber
SamplesAbsolute Migration Distance(mm)
Relative Migration Distance
Molcular Weight (kd)
3Unknown #1
38 0.388 41.68
4Bacillus amylo-liquefaciens lysate
36 0.367 44.43
5Commercial α-amylase from Aspergillus oryzae 1st band
37 0.378 42.99
5Commercial α-amylase from Aspergillus oryzae 2nd Band
42 0.429 36.32
6Commercial α-amylase from Bacillus licheniformis
39 0.402 39.85
7Saliva 1st band
5.5 0.056 85.08
7Saliva 2nd band
39 0.402 39.85
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Tabashir ChowdhuryID: 211219136
8Commercial α-amylase from porcine pancreas
37 0.381 42.60
9Bacillus licheniformis lysate 1st band
8.5 0.088 80.90
9Bacillus licheniformis lysate first band
40 0.412 38.54
9Bacillus licheniformis lysate third band
42 0.433 35.80
The second part of the experiment involved western blotting to detect the different alpha
amylases using anti-alpha amylase antibodies specific for human and bacillus amylases.
Figure 3 illustrates the western blot that was performed on the SDS gel. The western blot
has the same sequence of samples as the original gel, with a broad range protein marker
NEB P7701 in lane 1 followed by the unknown sample 2 in lane 2. Lane 3 was occupied
by unknown sample 1; lane 4 contains the B. amyloliquefaciens lysate. Lane 5 contained
commercial alpha amylase from A. oryzae, while lane 6 contains the commercial alpha
amylase for B. licheniformis. Lane 7 consists of the saliva sample and lane 8 contains the
commercial alpha amylase from porcine pancreas. Lane 9 contains the crude lysate from
the B.licheniformis. Lane 10 was loaded with another prestained protein marker.
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Tabashir ChowdhuryID: 211219136
It can be observed from figure 3 that, the unknown sample 2 in lane 2 didn’t exhibit any
bands. No bands were observed in lanes 6 and 9 either. Lane 3 containing the unknown
sample 1 shows a narrow band indicating an interaction with the antibody. Lane 4 (B.
amyloliquefaciens lysate) exhibits a collection of faint bands and a thicker band, which
had migrated roughly the same distance as the band from lane 3. The commercial alpha
amylase from A. oryzae also shows a large number of bands that resemble a smear, with
a thick predominant band with the same migration distance as the bands from lanes 3 and
4. No bands were observed in lane 6 which contained the alpha amylase from B.
licheniformis, indicating that no antibody interaction had taken place. Lane 7 containing
the human saliva sample showed only one thin band at the same distance as the other
bands that were observed so far.
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Figure 3 The figure illustrates the western blot image performed on the polyacrylamide gel after separation of the proteins by SDS- PAGE. Lanes 1 and 10 show the Broad range marker NEB P7701 and pre-stained protein marker NEB P7711S respectively. Lanes 2 and 3 contain the unknown samples 2 and 1 respectively. Lanes 4 and 9 contain the two bacillus cell lysates B. amyloliquefaciens (4) and B. licheniformis (9). The commercial alpha amylases are present in lanes 5 (A. oryzae) lane 6 (B. licheniformis), and lane 8 (alpha amylase from porcine pancreas). Lane 7 contains the human saliva sample. The molecular weights of the known proteins in the broad range protein marker are listed next to the image.
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Lane 8, which contained the commercial porcine alpha amylase, also showed a large
number of bands and a predominant band that has a similar migration distance as the
other samples. No bands were observed in lane 9, which contained the B. licheniformis
crude extract.
The absence of a band means that no antibody antigen interaction has occurred. This
implies an absence of alpha amylase in those samples, or an alpha amylase that wasn’t
recognized by the primary anti-alpha amylase antibodies due to a lack of similarity in
amino acid sequences.
All the samples that had interacted with the primary antibodies show a predominant band
at the same absolute distance, indicating the presence of a protein of roughly the same
size in all the samples.
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Discussion
From the SDS-PAGE of the samples we were able to determine the molecular weights of
the proteins in each of our samples. The protein in lane 6 (alpha amylase from B.
licheniformis) had an estimated molecular weight of 39.85kd. The molecular weight of
the commercial alpha amylase from B. licheniformis (Sigma A4551) is 62 kd5. The
protein in lane 8 (alpha amylase from porcine pancreas) had a molecular weight of 42.60
kd. The estimated molecular weight of the commercial alpha amylase from porcine
pancreas (sigma A3176) is between 51-54 kd6. The protein in lane 6 (alpha amylase from
A. oryzae) was estimated to be 42.99kd. The sample in lane 6 also showed a second band,
which had a molecular weight of 36.32 kd. The molecular weight of the commercial
alpha amylase from A. oryzae was estimated to be 49 kd5. Several factors can affect the
migration of proteins and cause them to migrate at a slightly different rate than predicted
based solely on its Molecular Weight7. Incomplete reduction of the sample, often
characterized by the presence of multiple bands at and around the predicted size of the
protein. This could account for the presence of two bands in the sample containing the
alpha amylase from Aspergillus oryzae. Differences in SDS binding can also account for
differences in molecular weigh estimates. Unique amino acid sequences can cause each
protein to bind SDS with varying affinity. This difference in binding can cause significant
differences in the actual mobility of the protein compared to what is predicted7. Using an
inappropriate percentage of polyacrylamide gel also affects protein migration and
therefore its molecular weight. Finally the migration of Molecular Weight Markers is an
important factor affecting molecular weight estimation, some markers may not be optimal
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for predicting the mass of the protein in the sample and although, use of pre-stained
molecular weight markers is very helpful for assessing transfer of proteins in immunoblot
applications, pre-stained markers may migrate at sizes slightly different from predicted
due to the presence of attached dye molecules7. Any or all of these factors could have
affected the estimation of the molecular weights of the proteins in the sample that would
account for the differences in the calculated molecular weights and the molecular weight
standard.
The bands present in the unknown sample 1 had a molecular weight of 41.68kd and the
band in the saliva samples molecular weight was estimated to be 39.85kd. The other band
in the saliva sample had a much higher molecular weight of 85kd. This could be a
contaminant present in the sample. Proteins in both these samples have molecular
weights similar to the estimated molecular weights of the commercial alpha amylases.
This allows us to conclude that these proteins are most likely to be alpha amylases.
The crude extracts of the Bacillus amyloliquefaciens showed only one band with a
molecular weight of 44.43kd, and the Bacillus licheniformis crude extract showed 3
bands with molecular weights of 80.90kd, 35.80 kd, 38.54 kd respectively. The last two
bands are likely to be amylases as they correspond to the molecular weight of the
commercial alpha amylase from B. licheniformis that was estimated in this experiment.
Unknown sample no. 1 showed no bands in the SDS-PAGE, so it is most likely a DNA
sample.
The western blot analysis allows us to draw a more precise conclusion regarding the
identity of protein bands in each sample. If a protein in a sample interacts with the anti-
amylase antibodies, thus resulting in the formation of a colored band in the membrane, it
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Tabashir ChowdhuryID: 211219136
conclusively proves the presence of alpha amylase in that sample. The presence of
prominent bands with a similar migration distance also supports this assumption.
Therefore the bands observed in lanes 3(unknown sample 1), 4(crude extract of bacillus
amyloliquefaciens), 5(alpha amylase from A. oryzae) 7 (saliva sample) and lane 8 (alpha
amylase from porcine pancreas) indicate the presence of alpha amylase in all of these
samples. The Absence of a band in the B. licheniformis alpha amylase sample and the
B.licheniformis crude extract suggests that there is no antigen-antibody interaction. This
is probably because the alpha amylase for B.licheniformis is sequentially different from
the B. amyloliquefaciens alpha amylase, and this difference prevents the anti-alpha
amylase from B. amyloliquefaciens from interacting with the alpha amylase of B.
licheniformis.
The western blot analysis allows the detection and identification of a particular protein
with some certainty, which is not always possible from estimating the molecular weight
of a protein using SDS-PAGE.
Questions: pg 54
1. During SDS-PAGE all the proteins are denatured by heat, reducing agent and by
the detergent SDS. SDS also coats all the proteins with a negative charge. As a
result, all proteins are negatively charged according to their mass and they all
have a similar rod like structure. Therefore when a voltage is applied the proteins
migrate to the positive electrode on the basis of their molecular weights.
2. The new gel should have a lower concentration of polyacrylamide. Since the
proteins of interest were packed together at the top, it indicates that they are
proteins of large molecular weight. So to separate the larger proteins we need to
use a gel that has a lower concentration of acrylamide.
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3. SDS-PAGE can be used to determine the molecular weight of a protein, the purity
of protein in a protein sample, and it can also be used to estimate the relative
amounts of protein present in a sample.
Questions pg 60
1. Immunoblotting is used to detect a specific protein separated by SDS-PAGE,
using an antibody specific for that protein, which will interact with the protein
after it is transferred to a membrane where the protein-antibody interaction can
be detected directly by a tag (fluorescent or radiolabelled) on the antibody, or
indirectly via another antibody attached to an enzyme. Immunoblotting can
confirm the presence of a particular protein in a sample containing a collection of
proteins (cell lysate). It can also detect whether a particular protein of interest is
similar to another protein.
2. The commercial alpha amylase from B.licheniformis did not react with the
antibodies. This is because of differences is amino acid sequences between the
alpha amylases of B. amyloliquefaciens and B. licheniformis. The lack of
similarity prevents the primary antibody (anti-alpha amylase from B.
amyloliquefaciens) from recognizing and interacting with the alpha amylase from
B. licheniformis.
3. One of the limiting factors of western blotting is that often the antibody used
doesn’t interact with all the proteins of interest due to the highly specific nature
of the antibodies. As a result proteins that are slightly different in amino acid
sequence or structure may not always be detected through immunoblotting.
4. If only lower molecular weight bands were observed in the lanes with the
Bacillus cell crude extracts, it would indicate that the during the preparation of
the samples, the alpha amylase proteins were degraded into smaller fragments.
Some of these fragments had interacted with the primary antibody, however since
they had been degraged the bands had migrated a greater distance during the
SDS-PAGE, which is why the bands of low molecular weight can be observed.
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References
1. Thiel, T., Bissen, S., and Lyons, E.M. (2002) Biotechnology: DNA to Protein – A Laboratory Project in Molecular Biology. McGraw-Hill, New York, pp.45-60.
2. De Souza PM and de Oliveira e Magalhaes P. 2010. APPLICATION OF MICROBIAL alpha-AMYLASE IN INDUSTRY - A REVIEW. Brazilian J Microbiol 41(4): 850-61
3. Strobl S, Maskos K, Betz M, Wiegand G, Huber R, Gomis-Rueth FX, Glockshuber R. 1998. Crystal structure of yellow meal worm alpha-amylase at 1.64 a resolution. J Mol Biol 278(3): 617-28.
4. Brzozowski, A. M. & Davies, G. J. (1997). Structure of Aspergillus oryzae alpha-amylase complexed with the inhibitor acarbose at 2x resolution. Biochemistry. 36, 10837-10845.
5. The Enzyme Handbook, Vol. 4, Schomburg, D., and Salzmann, M., Springer-Verlag (Berlin Heidelberg: 1991), EC 3.2.1.1, p. 7
6. Cozzone, P., et al., Characterization of Porcine Pancreatic Isoamylases Chemical and Physical Studies. Biochim. Biophys. Acta, 207(3), 490-504 (1970).
7. “Factors affecting migration of proteins in SDS-PAGE gels”. Novusbio. N.p., n.d. Web. 17 October 2011.
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