Tabashir Chowdhury ID: 211219136

BIOL3140
Advanced Biochemistry and Molecular Genetics Laboratory

Laboratory Report 2 Protein Analysis for alpha Amylase

TA: Mez

Date of Submission: 17/10/11

Tabashir Chowdhury ID: 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.

Tabashir Chowdhury ID: 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 bacteria . The main function of -amylases is to catalyze the breakdown of amylose and amylopectin through the hydrolysis of internal alpha-1, 4glycosidic linkages
3, 4 2

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 electrophoresis is ideal for analysis of proteins as it uses a detergent SDS and a reducing agent -mercaptoethanol to break down the native structure of the proteins and causing all proteins to have the same shape1. 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 Chowdhury ID: 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 product . 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 Chowdhury ID: 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) - 100l of TE (10mM Tris-HCl, pH 8, 1mM EDTA) is added to each of the 1.5ml tubes containing the bacterial culture. - 100l of dry beads were used which were added to the bacterial cell crude extracts. - 15l of each bacillus lysate is transferred to a new tube followed by 15l of 2X SDSPAGE loading buffer - 15l of saliva sample is transferred to a 1.5ml tube and 15l of 2X SDS-PAGE loading buffer is added to the sample. - 15l of each of the 3 commercial amylases and 2 unknown samples were added to 3 eppendorf tubes followed by 15 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.

Tabashir Chowdhury ID: 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.

Tabashir Chowdhury ID: 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.

Tabashir Chowdhury ID: 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 -amylase samples were run in lane 5, 6 and 8. With -amylase from porcine pancreas (Sigma A3176) in lane 8, -amylase from Aspergillus oryzae (Sigma A6211) in lane 5 and -amylase from B. licheniformis (Sigma A4551) in lane 6. A human saliva sample was run in lane7 and finally a second prestained 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 -amylases are further highlighted with on each band

Tabashir Chowdhury ID: 211219136 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.

Tabashir Chowdhury ID: 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 Distance migrated in gel Relative Mobility Rf weight in Kd (a) Phosphorylase b (97.4 kd) (b) Bovine serum albumin (66.2 kd) (c) ovalbumin (42.7 kd) (d) carbonic anhydrase (31kd) (e) trypsin inhibitor (21.5 kd) (f) lysozyme (14.4 kd) (mm) 8 18 27 37 55 64 0.08 0.1856 0.278 0.381 0.567 0.6598

120

100

Molecular Weight (kd)

80

60

40

20 y = -130.72x + 92.403 R = 0.8702 0 0.1 0.2 0.3 0.4 0.5 Relative Mobility Rf 0.6 0.7

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.

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Tabashir Chowdhury ID: 211219136

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 samples estimated molecular weight.

Lane Number 3 4 5 5 6 7 7 8 9 9 9

Samples Unknown #1 Bacillus amylo-liquefaciens lysate Commercial -amylase from Aspergillus oryzae 1st band Commercial -amylase from Aspergillus oryzae 2nd Band Commercial -amylase from Bacillus licheniformis Saliva 1st band Saliva 2nd band Commercial -amylase from porcine pancreas Bacillus licheniformis lysate 1st band Bacillus licheniformis lysate first band Bacillus licheniformis lysate third band

Absolute Migration Distance(mm) 38 36 37 42 39 5.5 39 37 8.5 40 42

Relative Migration Distance 0.388 0.367 0.378 0.429 0.402 0.056 0.402 0.381 0.088 0.412 0.433

Molcular Weight (kd) 41.68 44.43 42.99 36.32 39.85 85.08 39.85 42.60 80.90 38.54 35.80

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Tabashir Chowdhury ID: 211219136 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. It can be observed from figure 3 that, the unknown sample 2 in lane 2 didnt 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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Tabashir Chowdhury ID: 211219136

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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Tabashir Chowdhury ID: 211219136 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 wasnt 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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Tabashir Chowdhury ID: 211219136

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 kd . 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 kd . 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 kd . 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 Weight . 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 predicted . 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
7 7 5 6 5

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Tabashir Chowdhury ID: 211219136 not be optimal 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 molecules . 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 antiamylase antibodies, thus resulting in the formation of a colored band in the membrane, it
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Tabashir Chowdhury ID: 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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Tabashir Chowdhury ID: 211219136 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 doesnt 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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Tabashir Chowdhury ID: 211219136

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. 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 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. Brzozowski, A. M. & Davies, G. J. (1997). Structure of Aspergillus oryzae alphaamylase complexed with the inhibitor acarbose at 2x resolution. Biochemistry. 36, 10837-10845. The Enzyme Handbook, Vol. 4, Schomburg, D., and Salzmann, M., SpringerVerlag (Berlin Heidelberg: 1991), EC 3.2.1.1, p. 7 Cozzone, P., et al., Characterization of Porcine Pancreatic Isoamylases Chemical and Physical Studies. Biochim. Biophys. Acta, 207(3), 490-504 (1970). Factors affecting migration of proteins in SDS-PAGE gels. Novusbio. N.p., n.d. Web. 17 October 2011.

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