biol3140 lab 2

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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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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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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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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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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- 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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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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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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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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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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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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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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