1. EMG 9 and EMG 26 contain strain _lac-_(I- Z+ Y+) and strain _lac -_ (I+ Z- Y-)respectively.Three genes huddled together on the chromosome are required for two strains of _E.coli_ to utilize lactose.Consisting of three genes, namely, _lacZ_, _lacY_ and _lacA_, the _lac_ operon orderly handles these genes to code specific enzymes necessary for the metabolism of lactose. The genes _lacZ_, _lacY_ and _lacI_ would code for beta-galactosidase, galactosidase permease and _lac_ repressor respectively. Regulation of _lac_ operon is also tight and the operon's negative control is made possible by _lac_ repressor (Hill, 1996). With the presence of _lac_ repressor, _lac_ operon will be deactivated and will remain on this state unless acted upon by lactose. Consequently, transcribing the genes inside _lac_ operon will be prevented. _Lac_ operon only operates with the presence of lactose and absence of glucose. However, when both lactose and glucose are available, the latter is primarily preferred over metabolism. Lactose will only be cleaved into glucose and galactose with the aid of beta- galactosidase and its transportation inside the cells will be facilitated by galactoside permease (Hayes et al., 2010).
Inside EMG26, mutation can be noted on _lacZ_ gene. Production of beta-galactosidase will also not occur regardless of the current state of _lac_ operon, whether it is activated or not; or whether lactose is present or absent (Snyder, et al., 2007). Moreover, binding of _lac_ repressor to its operator is also apparent which in turn prevents the transcription of _lac_ genes through the inhibition of RNA polymerase. However, this inhibition may be counteracted with the presence of allolactase, an alternative lactose formation generated from rearrangements of beta-galactosidase. When allolactase binds, alteration in the conformation of repressor will occur in such way that it favors its dissociation with the operator (Hames, et al., 2000). Consequently, it stimulates the _lac_ operon, which results to the production of (_lac_ Y+) or galactosidase permease only. Galactoside permease is only produced since its genes do not undergo mutation. On the other hand, beta galactosidase is not produced when mutation of _lac_ Z gene occurs.
When it comes to EMG 9, the production of _lac_ repressor is not apparent due to _lacI_ gene mutation (_LacI-)_. Constant activation of _lac_ operon will happen regardless of lactose availability. In addition, transcription of _lac_ genes by RNA polymerase will occur. Therefore, it can be stated that galactosidase permease and beta-galactosidase will always be present since _lac_ operon remains activated and no mutation occurs with their genes (Pierce, 2007). Upon inspecting the gel used in this study, there were notable cellular variations between the protein pattern and the content of two _E. coli_ strains, EMG 26 and EMG 9. The identified difference between the two strains is primarily on the ability to manufacture beta-galactosidase protein, and this is apparent on the EMG 9 strain. Since EMG 9 can produce beta-galactosidase proteins, it is possible that bands are present on the eighth and ninth lane. As presented in Figure 2, two bands are identified to have high distinction and intensity on the topmost surface of the gel. In the sixth and seventh lane, where EMG 26 strain of _E. coli_ is cultured, no bands are discovered. This can be explained by the inability of this strain to produce beta-galactosidase proteins secondary to alteration of _lacZ_ gene.
Although Figure 2 reflects that two bands are present on the eighth and ninth lane, these types of bands do not correspond with beta-galactosidase protein. Rather, these two bands are similar to those bands present in lane 10. In this lane, numerous amounts of molecular markers which consist of seven proteins are laden. One of these proteins is beta-galactosidase, and this was regarded as the first band in the gel. Moreover, this type of protein made up the 116 kDa weight as shown in Figure 3. This identified weight is attributed to the characteristic of beta-galactosidase to be denatured by sodium dodecyl sulfate (SDS). In its original state, beta-galactosidase has a molecular weight of 464 kDa. Considering that this protein consists of four complex subunits, its exposure to the denaturing activity of sodium dodecyl sulfate can equally divide beta-galactosidase into four parts, each of which has a molecular weight of 116 kDa. Moreover, exposure to polycramide gel electrophoresis will result to alignment of subunits. The subunit possessing a molecular weight of 116 kDa will be regarded as the first molecular marker in the band (Zhang et al., 2005).
As mentioned previously, the two identified bands in the eighth and ninth lane, where EMG 9 is cultured, have high intensity and distinction. This can be explicated by the mutated _lacI_ gene found within this strain of _E.coli_. When this gene mutates, synthesis of _lac_ repressor will not transpire and activation of _lac_ operon will take place. Consequently, the non-mutated _lacZ_ gene within the EMG 9 strain will be transcribed and translated. Beta-galactosidase will become abundant resulting to increased intensity of bands. Therefore, it can be stated that there is direct relationship between protein abundance and intensity of the bands. Protein abundance will result to high band intensity (Hill, 1996).
The EMG 26 strain cultured on the sixth and seventh lane is a competent producer of _lacI_ gene. This gene produces _lac_ repressor proteins and such ability is absent on EMG 9. Therefore, the bands identified on the sixth and seventh lane are attributed to _lac_ repressor proteins. Conversely, these bands are absent on the eighth and ninth lane where EMG 9 is cultured. Just like beta-galactosidase, _lac_ repressor protein is a tetramer and its molecular weight at this state is 150 kDa. It can also be denatured by sodium dodecyl sulfate and such process would divide this protein into four equal subunits, each of which has a molecular weight of 37.5 kDa (Zhang et al., 2005). It can therefore be stated that these divided bands should be apparent between the third and fourth molecular markers as found in Figure 2. However, establishing the identity of its composition, whether it is made up of _lac_ repressor proteins or not, is difficult. This is the difference between the two _E.coli_ strains and such distinction can be visualized better with the enhancement of gel resolution.
Despite the differences identified, the other bands present on both cultures of EMG 26 and EMG 9 correspond to all cellular proteins. In addition, the positioning, amount, and type of these proteins are quite the same since they are both cultures of _E.coli_.
2. One of the most widely used methods for measuring and identifying proteins dissociated by PAGE or polyacramide gel electrophoresis is coomassie blue staining. This method marks all the proteins present in the gel by binding to the hydrophobic structures found in protein backbone. However, an acidic medium must be used in coomasssie blue staining to enhance the attraction of electrostatic and Van der Waals forces. These forces generate protein-dye complexes by binding together the building blocks of protein and the molecules of stain. Coomassie blue staining also has protein-dye ratio which contributes to its ability to dye proteins intensely. For every milligram of protein, commasie blue can bind around 1.2 to 1.4 milligrams of dye. The intensity of staining is also affected by the amount of certain amino acids found in the proteins since commassie blue can bind these proteins hydrophobically or ionically. These proteins would include arginine, histidine and lysine. Therefore, the abundance of these amino acids (histidine, arginine and lysine) on a protein would result to greater intensity on the stain produced by coomassie blue (Roe, 2001).
However, the downfall of using this kind of staining is insensitivity. As compared with fluorescence and silver staining, coomassie blue staining has low protein detection rate which is 30 to 100 nanograms only. In addition, this staining method cannot measure the amount of protein precisely. Difficulties with protein counting are attributed to the staining and destaining process utilized in this method. Previously dyed proteins by commassie blue staining need to be destained which consequently reduces the amount of dye attached to them (Roe, 2001).
3. Previously, it has been established that the abundance of protein is directly proportional to its intensity and thickness. When there are high amounts of protein, there are also notable increase in thickness and intensity of bands. Nevertheless, band thickness and intensity are affected by several factors. In the study conducted, the factor that affects the intensity and thickness of the bands is the amount of _E.coli_ strain used. Varying amounts of EMG 26 and EMG 9 strain are placed on each tube. The first and sixth tubes contain 10 microliters of EMG 26 and the same amount of EMG 9 strain is placed on the third and eighth tubes. On the other hand, 20 microliters of EMG 26 strain is used on the second and seventh tubes, tubes four and nine also have 20 microliters but of different strain, which is EMG 9. Although they have the same amount of commassie blue stain, differences in the thickness and intensity of bands are expected.
It is therefore expected that abundant proteins would result to bands will greater thickness and intensities. On the other hand, the presence of amino acids such as histidine, arginine and lysine on proteins would result to greater intensity on the staining of coomassie blue.
4. In this experiment, the cultures of _E.coli_ strain, EMG 26 and EMG 9, undergo protein extraction. The extracted proteins are subjected to polyacylamide gel electrophoresis and sodium docecyl sulfate (SDS). The SDS is a denaturing detergent, which divides proteins into smaller units in such a way that they cannot bind again. This is also used to identify the polypeptide composition of each protein complexes. Aside from identification, the SDS also coats each polypeptide with negative charges which allow movement of these particles towards the anode. It also coats the inherent charges of each subunit causing molecular-weight-based rather than charge-based electrophoresis. This results to rapid migration of polypeptides with low molecular weights as compared to those polypeptides with larger molecular weights (Nolden et al., 2007).
The gel is also exposed to coomassie blue staining and nitrocellulose paper. Coomassie blue is used on one side of the gel to provide better visualization of polypeptides under the Gel Imaging System. The other side utilized nitrocellulose paper by blotting it on the gel to identify specific antibodies on beta-galactosidase. Antibody detection is done using monoclonal antibody of anti-beta-galactosidase, an agent distilled from the ascitic fluid of a mouse with hybidroma (Bazin, 1990). Since this monoclonal antibody can detect beta-galactosidase proteins of E.coli, it will attach itself with a high specifity to portions of previously blotted nitrocellulose paper where beta-galactosidase is present.
Visualization of beta-galactosidase protein in nitrocellulose paper necessitates another form of antibody which is called the anti-mouse immunoglobulin-G alkaline phosphate conjugate. It can detect immunoglobulin-G mouse antibodies that have formed complexes with beta-galactosidase proteins and covalent linkages with enzymes of alkaline phosphatase. This is regarded as indirect method of beta-galactosidase protein detection since its amount is determined by the reaction of alkaline phosphatase to the added 5-bromo-4-chloro-3-indolylphosphate (BCIP) substrate. Therefore, complexes of alkaline phosphatases and protein antibodies exist whenever beta-galactosidase is present (Jowett, 2009).
The presence of the BCIP is also necessary for color detection especially with the supplementation of nitro blue tetrazolium (NBT). Phosphorylated compounds, which are products of BCIP substrate, become illuminiscent if they are stripped off of their phosphate group. Such stripping is possible with the cleaving action of alkaline phosphatase. Adding NBT will allow detection of illuminiscented products of BCIP substrate which appears as purple-gray in the nitrocellulose paper (Eisenthal et al., 2002). The enzyme will then augment the signal which detects the presence of beta-galactosidase. As beta-galactosidase increases in number, so does the intensity of the enzymatic activity. Thus, the intensity of band color is directly proportional to the quantity of beta- proteins present (Eisenthal et al., 2002).
5. Within the immune system are proteins that activate in response to the presence of foreign material. These are the antibodies. However, their responses are mediated with an antigen, which contributes to the ability of antibodies to react only with a certain substance and not with other substances (Orazi et al., 2007).Because of their ability to differentiate one molecular structure to the other, antibodies are utilized to quantify, purify and evaluate biological molecules and to treat medical conditions. Interestingly, the reaction with these antigens is necessary in the production of several types of antibodies. One of them is the monoclonal antibody. (Orazi et al., 2007) As the name suggests, this antibody consists only of a single antibody which reacts to a specific antigen. It has a single isotope of immunoglobulin and affinity and is utilized to extract antibodies from proteins that cannot be sequenced or purified. They are also used on proteins that are low in quantity (Orazi et al., 2007). Since monoclonal antibodies are monoepitopic, detecting locations for the functioning of proteins are feasible. Nevertheless, becoming monoepitopic makes them prone to protein alterations such as fixation with aldehydes, denaturation with the SDS, and modifications after its transition (Orazi et al., 2007).
Polyclonal antibody is another type of antibody produced with reaction to an antigen. It contains various molecules of antibodies which act on a single antigen and respond regardless of the antigen present. If monoclonal antibody has monoepitopic specificity, polyclonal antibody has polyepitopic specificity (Buchwalow et al., 2007).This distinctive feature of polyclonal antibody facilitates the recognition of epitopes even if they are fixated by aldehydes or denaturated by the SDS since not all of epitopes are obliterated after these processes(Buchwalow et al., 2007). As compared to the single affinity exhibited by monoclonal antibodies, affinities of polyclonal antibodies for a single antigen are varied. The minor disparities found on the amino acids of these antibodies allow them to act variedly on the same antigens. Polyclonal antibodies are also favored over monoclonal antibodies since they provide an easy, quick and economical method of antibody production. It is very different when one produces monoclonal antibodies since they are costly, protracted, and necessitate extensive skills and equipment to culture cells (Buchwalow et al., 2007).
6. The purple-gray color found on the nitrocellulose paper with anti-beta-galactosidase monoclonal antibody signifies the presence of beta-galactosidase proteins. The color is produced after the BCIP substrate and the NBT reacts with complexes formed between protein antibody and alkaline phosphatase. Therefore, it is anticipated that the purple-gray color will be evident on the third and fourth lanes which contain 10 microliters and 20 microliters of cultured EMG 9 E.coli strain, respectively. This is possible since the EMG9 strain consists of strain _lac -_ (I- Z+ Y+). Since this strain has a mutated _lacI_ gene, it is impossible to synthesize _lac_ repressors. Therefore, _lac_ operon will be activated regardless of the presence of allolactose. Constant translation and transcription of _lac_ genes will occur, which results to continuous production of galactosidase permease and beta-galactosidase. Figure 2 shows the presence of beta-galactosidase proteins (molecular weight of 116 kDa) on the first bands of third and fourth lanes as color purple-gray. This is possible since the molecular marker is made up of beta-galactosidase proteins. It is also expected that the fifth lane will have proteins of beta-galactosidase since this lane contains beta-galactosidase proteins.
On the other hand, the color identified is not expected on the first and second lanes since they contain 10 and 20 microliters of EMG 26 E.coli strain, respectively. This can be explained by the mutated _lacZ_ gene found on strain _lac -_ (I+ Z- Y-). The mutation of _lacZ_ gene will prevent the formation of beta-galactosidase proteins regardless of lactose availability. Since no mutation occurs on _lacI_ gene, production of _lac_ repressor will not occur. With absence of _lac_ repressor, _lac_ operator will be deactivated. On the other hand, the presence of lactose will result to activation of the _lac_ operator. _Lac_ operon will also be turned on if an inducer called allolactose is present. If ever operon is turned on, production of galactosidase permease will occur since _lacI_ gene is not mutated. Beta-galactosidase protein, on the other hand, will not be present because _lacZ_ gene is mutated.
Upon inspecting the third, fourth and fifth lanes, it is noted that beta-galactosidase proteins, with a molecular weight of 116 kDa, is at the topmost part of the gel. On the lanes where beta-lactoside is available, purple-color is present due to the formation of complexes, which result to the reaction between protein-antibody and anti-body alkaline phosphatase. On those lanes where beta-lactoside is unavailable, no purple-gray color can be noted. Nevertheless, false positive results may occur. This means that purple-gray color is present despite beta-lactoside unavailability.
7. Figure 2 illustrates the bands detected on the third and fourth lanes of nitrocellulose paper blotted with protein content of EMG 9 _E.coli_ positive strain. This strain is a competent producer of beta-galactosidase and such ability is attributed to the _lac -_ (I- Z+ Y+). Within this strain, mutated _lacI_ gene and non-mutated _lacZ_ gene can be found. Such orientation results to the absence of _lac_ repressor which in turn closes the _lac_ operon.
Further examination of the nitrocellulose paper presented in Figure 2 reveals the presence of multiple bands and the absence of single bands. Normally, single bands become evident if the antibody successfully reacts with beta-galactosidase protein resulting to the formation of complexes (Pierce, 2007). Consequently, the color purple-gray will appear on the topmost part of the gel in line with the first bands to signify beta-galactosidase proteins. If single bands are absent, there must be factors that affect these reactions. Elucidating the western blot process will allow clarification of the identified phenomenon. Augmenting the sensitivity of western blot requires treatment of nitrocellulose paper with TBST + 1% BSA. This is done to prevent fixation of antibodies on locations where nonspecific protein binding can take place. This is also the aim of the experiment. When the nitrocellulose paper is not properly treated with the TBST + 1% BSA, there will be insufficient blocking which results in multiple bands. Moreover, excessive protein binding sites will be present allowing other antibodies to bind. Thus, if anti-beta-galactosidase monoclonal ntibody, the primary antibody, binds to nonspecific protein binding sites, the secondary antibody which is the anti-mouse-immunoglobulin-G alkaline phosphatase conjugate will also bind. This causes the formation of protein complexes, which can produce purple-gray color upon reacting with the added NBT and BCIP substrate. False positive results will occur since beta-galactosidases proteins are not involved in the whole process.
False positive results can also occur if monoclonal antibodies cross-react with non-target proteins and non-specific proteins. Epitopes have various forms and they can be conformational or linear in nature. Epitopes become conformational if residues of amino acid fold in such a way that it is far from the protein sequence. Imitation of linear epitopes is also possible with the aid of simple sequences of peptides. Such imitation can also happen with conformational epitopes which generate the so-called mimotopes (Westwood et al., 2007). Although nitrocellulose paper is properly blocked and the beta-galactosidase proteins have successfully attached to specific protein binding sites, cross-reaction can still occur. If parts of epitope sequences are present on the mimotopes and linear epitopes that have cross-reacted, there is a great tendency that these proteins can be identified by antibodies found on the nitrocellulose paper. Reaction and binding can take place on the primary antibody which in turn allows binding with the secondary antibody. Consequently, it forms complexes and reaction with the NBT, and the BCIP will result to purple-gray color.
False positive results also happen if the proteolytic enzymes found on the cultures of _E.coli_ have degraded beta-galactosidase proteins. Even though beta-galactosidase proteins are degraded, epitopes are still present and they still contain the target epitope (Balsubramanian et al., 2004). Therefore, recognition of the primary and secondary antibodies is still possible. They can also form complexes that produce small-fragmented purple-gray color upon reacting with the added NBT and BCIP. Since the fragmented epitopes from degraded beta-galactosidase have less friction, they can move further towards the anode. This process explains why multiple bands are present on the lower ends of the third and fourth lanes of nitrocellulose paper (see Figure 2).
LABORATORY MANUAL QUESTIONS
1. The emergence of band on the gel stained with coomassie blue denoted that proteins are present. These proteins were acquired from the extraction process performed on both cultures of _E.coli_ strains, EMG26 and EMG 9, which were later subjected to sodium docecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, inspection of the topmost band on the eighth and the ninth lane revealed the presence of beta-galactosidase, the only protein that can be detected in the gel stained by coomassie blue.
2. Inspection of the nitrocellulose paper revealed the presence of multiple bands and this can be explained by several reasons. First, the appearance of multiple bands may have something to do with nitrocellulose paper's blocking process. Insufficient blocking of nonspecific proteins will allow antibodies to bind on these sites and this will generate a false positive result.
The proteolytic activity of enzymes present in the cultures of E.coli may also explain why multiple bands are present on the nitrocellulose paper. Isolation of proteins on E.coli cultures allowed enzymes to degrade beta-galactosidase into smaller fragments. Although they are degraded, the resulting fragments still contain an epitote that can be still be detected by antibodies. Once detected, antibodies will bind to them and this would result to another false positive result.
Monoclonal antibodies are capable of cross-reaction and this process can explain the presence of multiple bands. These antibodies are capable of binding with non specific proteins as long as they contain partial sequence of the epitote protein they really target. Once binding occurs, there will be false positive results.
Multiple bands can be detected on the third, fourth and fifth lane. This became possible since the third and the fourth lane contains the culture of EMG 9 _E.coli_ strain. On the other hand, the fifth lane contains beta-galactosidase, one of the seven proteins found on its molecular marker. Furthermore, beta-galactosidase with a molecular weight of 116kDa represents the topmost bands on these three lanes.
Nevertheless, multiple bands are not present on the first and second lane since they both contain cultures of EMG26. In this type of strain, the _lacZ_ gene on its _lac_ operon is mutated. This results to absence on the production of beta-galactosidase. With the absence of beta-galactosidase, no antibody-binding will occur.
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