Gel Electrophoresis and the Action of Alkaline Phosphatase

jelly cataphoresis and the effect of Alkaline PhosphataseIntroductionIn this practicable, two common proficiencys found in clinical laboratories be performed. The showtime technique is c whollyed gel cataphoresis and the import is an enzyme exercise assay. dielectrolysis is a method that expends an electrical field to divorce proteins by molecular size. In this case, the protein extracted in interoperable 1 and an mystical protein be sepa lay outd and lose itd using a polyacrylamide gel electrophoresis (PAGE). Electrophoresis is a popular and widely employ analytical technique in research, it apprise be used for a variety of applications but its just about widespread use is the separation of proteins to and soce analyse and purify them. The technique has with child(p)ly evolved over the years since the instrumentation, buffer systems and visualization techniques sw eitherow on the whole been rapidly improving. This has helped to create several(predicate) prot ein electrophoresis techniques such as isoelectric nidus (IEF) or electrophoretic transfer (commonly k outrightn as Blotting) which are great tools used in modern research methods (facebook knave).The consequence experiment is an enzyme reckon reply experiment that uses alkaline phosphatase (ALP). Where the enzyme activity of a commercialized messagely on tap(predicate) purified form of ALP is compared to the ALP activity of the prison cell lysate prepared in practical 1.A chemical substance answer drift brush off be influenced by the social movement of enzymes, these proteins tummy turn a chemical reaction by sour the activation energy of the reaction. They grass do this all while be unchanged, making them a perfect locoweeddidate for a marker to monitor a chemical reaction rate. These reactions are found in all living organisms and naturally occur in metabolic pathways for example. The activity of an enzyme screwing be altered by a change in the pH, the ducki ng of the enzyme or the substrate, the temperature and by the presence of inhibitors. By controlling these changes the activity of an enzyme smoke be reliably monitored. Enzymes are very specific to their corresponding substrate. When an enzyme is miscellaneous with its specific substrate in vitro, under optimum conditions, the substrate go away bind to the active site of the enzyme to form the enzyme-substrate complex at a steady rate. Thus, until the substrate is used up or the enzyme begins to denature or the complex formed changes the reaction conditions. By monitoring the harvest-tides of a chemical reaction, we trick analyse the rate of production of enzyme-substrate complexes. In this experiment, ALP is the enzyme that speed ups up the hydrolysis reaction that occurs to p-nitrophenyl phosphate to form p-nitrophenol. ALP is mainly found in the liver, bone, kidney but it is alike produced by the cells in the small intestine. The CACO-2 cells used in practical 1 have ver y similar traits to cells found in the small intestine, therefore, the ALP activity in the extract force out be measuring rodd. By monitoring the movement of the reaction during various time points, the activity of ALP earth-closet be determined.ElectrophoresisMaterialspipets and tipsDeionized wetElectrophoresis polyacrylamide gelElectrophoresis machine carrell lysate (practical 1)Protein XColour prestained Protein standardLaemlii buffer NuPAGE LDS render buffer 4x lot1658555 receptive on the 27/07/2015Coo toiletie blueRunning buffer regularitysFirstly, a freight warning containing the cell lysate prepared in practical 1 was make by adding 2l of cell lysate, 3l of water and 5l of laemlii buffer into an Eppendorf tube. A second loading warning containing protein x was prepared by adding 10l of protein x to 10l of laemlii buffer into an Eppendorf tube. The samples were wherefore added to a heated bath for 2 minutes.During this time, the polyacrylamide gel was opened and th e comb and tape were gently take. The electrophoresis cell was then assembled before filling the inner and outer buffer house with provided running buffer. The inner bedroom had more buffer than the outer chamber to totally incubate the gel in the buffer.10l of the protein x sample, 3l of the ladder and 14l of our cell lysate sample were then loaded onto the gel in different surface by carefully inserting them using a pipette with slender tips. Once the apparatus was correctly assembled, the electrophoresis cell was connected to the index supply and the electrophoresis was performed at 150mv for 1 and a half hours. later on completion of the migration of the bands, the power supply was turned off and the electrical conk outs were disconnected. The gel cassette was then removed and the gel was gently transferred by floating it off the plate. The gel was then stained using Coomassie blue for an hour before transferring it to water. A register of the gel was then taken for pul l ahead interpretation.ResultsBy measuring the migration maintain travelled by the bands of proteins of known molecular weight, we can plot a standard curve of the distance travelled versus the molecular weight bow 1. Standard bands migration distance versus fragmentise sizeStandard distance travelled (cm)Ladder fragment size (kDa)22452.71903.51354.5 one C5.6807.1588.54610.33211.62512.62213.41714.111 common fig 3. Standard curve of the migration distance versus ladder fragment size of the protein standardThis produces an equation that can be used to measure the sizes of the bands produced by the protein x sample.Table 2. Relative size of protein x components. bind numberProtein x Sample distance travelled (cm)Protein x sexual congress size proteins (kDa)11.4232.3422.3189.7533.4148.1546.770.5DiscussionThe bands notice in figure 1 are composed of proteins of the same size. The proteins are loaded in the negative end of the gel since they are negatively recoild, as the electrophor esis reaction is occurring, the negative current will affect the samples towards the positive end. The little samples will travel fast-breaking and thus further through the gel whereas bigger sized proteins will tend to emigrate less. This difference in migration is payable to the structure of the gel, it has fine filaments that can be re confronted as a mesh. The density of the gel is dependent on the concentration. The smaller proteins will find it easier to travel through the mesh whereas the larger molecules will move much more slowly (facebook page).Also, we can keep abreast that some bands are darker than others, this is because the darker bands have a higher concentration of a occasionicular protein of the same size. We can estimate the molecular weight of the proteins by comparability the migration distances of the bands against the standard seen in well 1 (see figure 1). We can too observe the number of different protein sizes that are present in our samples by cou nting the number of bands. For example, our sample of protein x contains 4 macroscopical bands, meaning there are 4 protein groups in protein-x.The almost momentous band in the protein x separation is the in conclusion band containing the smaller fragments of protein. This band is estimated to have proteins of about 70.5 kDa. This band can besides be seen in the electrophoresis separation of the cell lysate prepared in practical 1. The band is seen in both samples because it is the band containing albumin. Albumin is the most large protein in the blood. It has a molecular mass of between 65-75 kDa which encompasses the estimated 70.5kDa of the proteins found in the bands calculated earlier (all about albumin, theodore Peters).In this practical, the use of beta-mercaptoethanol (BME) is used in combination with the sample buffer prior gel electrophoresis. It is activated by heating the sample and permits the successful migration of the subunits of the proteins during electrophore sis. It works by independently separating them on the SDS-PAGE. It completely denatures the disulphide bonds within the subunits to let the peptides freely migrate according to their chain length. By overcoming forms of tertiary protein folding and lysing oligomeric subunits, the influence of secondary structures is minimized. Sodium dodecyl sulphate (SDS) is also used during the experiment, as discussed in practical 1, this substance is an anionic detergent and is used during electrophoresis to linearize and promote the negative charge of the proteins prior to gel electrophoresis. The result of this is the even distribution of charge throughout the protein to help separate the protein fragments according to their size (Detergent binding explains anomalous SDS page migration of membrane proteins).To stain the proteins in this practical, a Coomassie stain was used. This protein stain is the most common anionic protein dye. It is popular because it stains most proteins and has grea t advantages such as good quantitative linearity, good use in identification during mass spectrometry and short staining time, for example. other(a) dyes can be used in gel electrophoresis such as silver stains. These stains have very high sensitivity, but unlike Coomassie Blue, they offer a glare linear dynamic range and are usually complex, therefore the protocols are time-consuming. Also, they do not offer sufficient reproducibility for quantitative analysis. Other type of stains that are commonly used are fluorescent stains. These stains also offer high sensitivity but, unlike silver stains, have a wider linear dynamic range and are simple to use and robust. The hurt is that they are more expensive to use and require specific tomography equipment such as scanners to view the gel (facebook page).The electrophoresis technique is now a routinely used method used in clinical laboratories to screen for protein abnormalities using samples of blood serum, urine or cerebral spinal unruffled and can analyse specific proteins such as enzymes (ALP or LDH), lipoproteins or haemoglobin. These techniques are evaluated visually for the presence of abnormal protein bands and can also be quantitively measured to determine the concentration of the bands.In a normal serum protein electrophoresis, 5 distinct bands appear on the gel the highest band contains albumin, followed by smaller bands containing alpha-1 globulins, alpha 2 globulins, beta globulins and finally gamma globulins. Analysing these bands can determine if abnormalities are present in the major proteins found in the body and can therefore be a valuable symptomatic tool. For example, changes in the zone containing the albumin band can help key out various abnormalities such as bisalbuminemia (2 bands instead of 1) and hyperalbuminemia. Significant changes in concentrations of other bands of the serum protein electrophoresis can easily help determine some different pathological disorders. The most common use of serum protein electrophoresis is for the diagnosis of multiple myeloma. An abnormal peak in a domain of the gamma globulin area can indicate a monoclonal gammopathy. monoclonal gammopathies have been shown to be associated with an anomalous clonal process that can lead to the development of cancerous tumours such as multiple myeloma (Patterns of serum protein electrophoresis, our acquaintance at King Hussein Medical Center, Jordan).Another common use of electrophoresis in a clinical laboratory is lipoprotein electrophoresis. This method determines the concentrations of different lipoproteins such as LDL. High plasma levels of LDL have been associated with vivid myocardial infarction and other heart related diseases.ConclusionGel electrophoresis is used to separate proteins according to their sizes by migrating them through a gel using an electric gradient. The smaller proteins will migrate faster and further than larger sized proteins due to the structure of the gel. This technique can be used in various clinical settings, for example, to analyse lipoproteins or serum proteins to help diagnosis various conditions.Enzyme activity of Alkaline PhosphataseMaterialsPipette and tips96 well plate mercantile ALP carrell lysate from practical 1 cellular phone lysate providedLysis bufferPara nitrophenol phosphate (PNP)3M NaOH (stop solution)Plate readerMethodThe experiment was performed in different steps to minimize potential errors due to timing issues. The first was the monitoring of the commercial ALP enzyme reaction rate in combination with the snowy test.This was done by adding 100l of the commercial ALP into 6 wells of the same line. The enzyme substrate Paranitrophenol phosphate was then added to all the wells as fast as possible to maintain a homogenous reaction in all the wells. Prior to the addition of the enzyme and the substrate, 50l of the stop solution (NaOH) was added to the first well to provide an sign reaction rate of 0s. 50 l of stop sol ution was then added to the other wells at a 3-minute interval until the final sixth well (t=15min). The plate was then read at 410nm and the results were collected. During this time, a blank test was performed by using the same method. The only difference was that the wells only contained 200 l of enzyme substrate and therefore no enzyme.After this was performed, an enzyme rate reaction for the provided cell lysate was done. Firstly, a stock solution of 700 l was done by adding 350 l cell lysate with 350 l of buffer. 100 l of the cell lysate stock solution was added to 6 wells. The first well also contained 50 l of the stop solution as mentioned earlier. 100 l of enzyme substrate was then added to all the wells as fast as possible. After 3 minutes, 50 l of the stop solution was then added to the second well, followed by the third 3 minutes later, and so on until the last well. The plate was then read at 410 nm on the plate reader.The final enzyme reaction contained the cell lysate prepared in practical 1. Firstly, a 700 l stock solution of cell lysate was done by adding 175 l of the cell lysate created in practical 1 to 525 l of lysis buffer. 100 l of the cell lysate stock solution was added to 6 wells. The first contained 50 l of stop solution as mentioned earlier. 100 l of enzyme substrate was then added to all the wells as fast as possible. After 3 minutes, 50 l of stop solution was added to the second well, followed by the third 3 minutes later, and so on until the last well. The plate was then read at 410nm on the plate reader. This experiment was done twice to provide duplicates.Table 3. 96 well plate distribution (time (t) in minutes) 1 (t=0)2 (t=3)3 (t=6)4 (t=9)5 (t=12)6 (t=15)A uncloudedBLANKBLANKBLANKBLANKBLANKBCCommercialALPCommercialALPCommercialALPCommercialALPCommercialALPCommercialALPDE applicatory 1 Cell lysate applicatory 1 Cell lysate practicable 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysateFGPractical 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysatePractical 1 Cell lysateHProvided Cell lysateProvided Cell lysateProvided Cell lysateProvided Cell lysateProvided Cell lysateProvided Cell lysateResultsTable 4. 96 well plate absorbance (410nm) results1 (t=0)2 (t=3)3 (t=6)4 (t=9)5 (t=12)6 (t=15)A0.2840.3030.2880.3440.2940.290BC0.2770.3550.4330.5040.5820.674DE0.6620.3960.4830.6350.6851.131FG0.3300.5440.4870.5630.6140.708H0.3290.5450.7400.8140.9150.967By using these absorbance, we can plot a graph of the absorbance versus the time for the various tested samples to analyse and compare them. Note that the results from well E1 and G2 have been omitted due to the errors occurred during pipetting (E1 well is t=0 but absorbance is abnormally high and G2 absorbance is abnormally high). Fortunately, these wells were part of a duplicate so the other result from the sample was kept.Figure 4. Graph of the absorbance over time of the commercial A LP, the cell lysate from practical 1 and the provided cell lysate.The activity of an enzyme can be measured by ascertain the rate of the formation of the product or the rate at which the substrate is used up. The rate of the reaction decreases when the substrate is being used up, therefore, the rate must be measured during the period when the formation of the product or decrease in substrate is linear with time. The rate of a reaction at time 0 is called the sign linear reaction rate (V=0min). By using the polynomial equations for each curve, an initial rate can be determined where V0=A410min-1. In other words, the value (b) in take care of x in the quadratic equation y=ax2+bx+c is the initial rate of the reaction ( youtube vid).Assuming that 0.1 mM of the solution of the reaction product produces an absorbance of 1, we can determine the enzyme rate as shown below.Table 5. Initial grade for each sampleSampleInitial rate (Abs/min)Enzyme rate (mM/Min)Practical 1 lysate0.10590.0105 9Blank0.03360.00336Commercial ALP0.06950.00695Provided ALP0.27450.02745DiscussionBy using this technique, we can calculate how fast an enzyme can catalyse a reaction. In this case, we can compare the rate of reaction of the cell lysate, the provided ALP and the commercial ALP to the blank sample as shown belowCell lysate (0.0059/0.00336) = 1.756It can be said that the ALP present in the cell lysate from practical 1 sped up the reaction 1.756 times faster compared to the reaction without it.Commercial ALP (0.00695/0.00336) = 2.065It can be said that the commercial ALP sped up the reaction 2.065 times faster than without the commercial ALP.Provided ALP (0.02745/0.00336) = 8.17It can be said that the provided ALP sped up the reaction 8.17 times faster than without the provided ALP.ConclusionALP is a widely-used enzyme in our body, it removes phosphate groups by a process called dephosphorisation. Its activity can be measured in vitro by monitoring its activity during a chemical reactio n in controlled conditions. The experiment used different samples containing ALP to catalyse the reaction of p-nitrophenyl phosphate to form p-nitrophenol. In conclusion, the results confirmed that ALP can speed up a reaction and this acceleration was measured by comparing the rate of reaction compared to a blank sample.