Gel Electrophoresis and the Action of Alkaline Phosphatase

Gel Electrophoresis and the Action of Alkaline Phosphatase Introduction In this practical, two common techniques found in clinical laboratories are performed. The first technique is called gel electrophoresis and the second is an enzyme activity assay.      Ã‚   Electrophoresis is a method that uses an electrical field to separate proteins by molecular size. In this case, the protein extracted in practical 1 and an unknown protein are separated and analysed using a polyacrylamide gel electrophoresis (PAGE). Electrophoresis is a popular and widely used analytical technique in research, it can be used for a variety of applications but its most widespread use is the separation of proteins to then analyse and purify them. The technique has greatly evolved over the years since the instrumentation, buffer systems and visualization techniques have all been rapidly improving. This has helped to create different protein electrophoresis techniques such as isoelectric focusing (IEF) or electrophoretic transfer (commonly known as Blotting) which are great tools used in modern research methods (facebook page). The second experiment is an enzyme rate reaction experiment that uses alkaline phosphatase (ALP). Where the enzyme activity of a commercially available purified form of ALP is compared to the ALP activity of the cell lysate prepared in practical 1. A chemical reaction rate can be influenced by the presence of enzymes, these proteins can catalyse a chemical reaction by lowering the activation energy of the reaction. They can do this all while remaining unchanged, making them a perfect candidate 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 can be altered by a change in the pH, the concentration of the enzyme or the substrate, the temperature and by the presence of inhibitors. By controlling these changes the activity of an enzyme can be reliably monitored. Enzymes are very specific to their corresponding substrate. When an enzyme is mixed with its specific substrate in vitro, under optimum conditions, the substrate will 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 f ormed changes the reaction conditions. By monitoring the products of a chemical reaction, we can analyse the rate of production of enzyme-substrate complexes. In this experiment, ALP is the enzyme that speeds 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 also produced by the cells in the small intestine. The CACO-2 cells used in practical 1 have very similar traits to cells found in the small intestine, therefore, the ALP activity in the extract can be measured. By monitoring the course of the reaction during various time points, the activity of ALP can be determined. Electrophoresis Materials Pipettes and tips Deionized water Electrophoresis polyacrylamide gel Electrophoresis apparatus Cell lysate (practical 1) Protein X Colour prestained Protein standard Laemlii buffer: NuPAGE LDS sample buffer 4x lot#1658555 opened on the 27/07/2015 Coomassie blue Running buffer Methods Firstly, a loading sample containing the cell lysate prepared in practical 1 was made by adding 2 µl of cell lysate, 3 µl of water and 5 µl of laemlii buffer into an Eppendorf tube. A second loading sample containing protein x was prepared by adding 10 µl of protein x to 10 µl of laemlii buffer into an Eppendorf tube. The samples were then added to a heated bath for 2 minutes. During this time, the polyacrylamide gel was opened and the comb and tape were gently removed. The electrophoresis cell was then assembled before filling the inner and outer buffer chambers with provided running buffer. The inner chamber had more buffer than the outer chamber to totally incubate the gel in the buffer. 10 µl of the protein x sample, 3 µl of the ladder and 14 µl of our cell lysate sample were then loaded onto the gel in different wells by carefully inserting them using a pipette with slender tips. Once the apparatus was correctly assembled, the electrophoresis cell was connected to the power supply and the electrophoresis was performed at 150mv for 1 and a half hours. After completion of the migration of the bands, the power supply was turned off and the electrical leads 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 picture of the gel was then taken for further interpretation. Results By measuring the migration distance travelled by the bands of proteins of known molecular weight, we can plot a standard curve of the distance travelled versus the molecular weight: Table 1. Standard bands migration distance versus fragment size Standard distance travelled (cm) Ladder fragment size (kDa) 2 245 2.7 190 3.5 135 4.5 100 5.6 80 7.1 58 8.5 46 10.3 32 11.6 25 12.6 22 13.4 17 14.1 11 Figure 3. Standard curve of the migration distance versus ladder fragment size of the protein standard This 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. Band number Protein x Sample distance travelled (cm) Protein x relative size proteins (kDa) 1 1.4 232.34 2 2.3 189.75 3 3.4 148.15 4 6.7 70.5 Discussion The bands observed 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 charged, as the electrophoresis reaction is occurring, the negative current will push the samples towards the positive end. The smaller samples will travel faster and thus further through the gel whereas larger sized proteins will tend to migrate less. This difference in migration is due to the structure of the gel, it has fine filaments that can be represented 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 observe that some bands are darker than others, this is because the darker bands have a higher concentration of a particular protein of the same size. We can estimate the molecular weight of the proteins by comparing the migration distances of the bands against the standard seen in well 1 (see figure 1). We can also observe the number of different protein sizes that are present in our samples by counting the number of bands. For example, our sample of protein x contains 4 visible bands, meaning there are 4 protein groups in protein-x. The most significant band in the protein x separation is the last band containing the smaller fragments of protein. This band is estimated to have proteins of about 70.5 kDa. This band can also 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 abundant 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 electrophoresis. 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 bi nding 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 great advantages such as good quantitative linearity, good use in identification during mass spectrometry and short staining times, for example. Other dyes can be used in gel electrophoresis such as silver stains. These stains have very high sensitivity, but unlike Coomassie Blue, they offer a lower 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 disadvantage is that they are more expensive to use and require specific imaging 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 serum, urine or cerebral spinal fluid 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 diagnostic tool. For example, changes in the zone containing the albumin band can help diagnose 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 many different pathological disorders. The most common use of serum protein electrophoresis is for the diagnosis of multiple myeloma. An abnormal peak in a region 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 experience 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 acute myocardial infarction and other heart related diseases. Conclusion Gel 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 Phosphatase Materials Pipette and tips 96 well plate Commercial ALP Cell lysate from practical 1 Cell lysate provided Lysis buffer Para nitrophenol phosphate (PNP) 3M NaOH (stop solution) Plate reader Method The 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 blank test. This was done by adding 100 µl 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, 50 µl of the stop solution (NaOH) was added to the first well to provide an initial reaction rate of 0s. 50  µl of stop solution was then added to the other wells at a 3-minute interval until the final 6th 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  µl 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  µl was done by adding 350  µl cell lysate with 350  µl of buffer. 100  µl of the cell lysate stock solution was added to 6 wells. The first well also contained 50  µl of the stop solution as mentioned earlier. 100  µl of enzyme substrate was then added to all the wells as fast as possible. After 3 minutes, 50  µl 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  µl stock solution of cell lysate was done by adding 175  µl of the cell lysate created in practical 1 to 525  µl of lysis buffer. 100  µl of the cell lysate stock solution was added to 6 wells. The first contained 50  µl of stop solution as mentioned earlier. 100  µl of enzyme substrate was then added to all the wells as fast as possible. After 3 minutes, 50  µl 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 BLANK BLANK BLANK BLANK BLANK BLANK B C Commercial ALP Commercial ALP Commercial ALP Commercial ALP Commercial ALP Commercial ALP D E Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate F G Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate Practical 1 Cell lysate H Provided Cell lysate Provided Cell lysate Provided Cell lysate Provided Cell lysate Provided Cell lysate Provided Cell lysate Results Table 4. 96 well plate absorbance (410nm) results 1 (t=0) 2 (t=3) 3 (t=6) 4 (t=9) 5 (t=12) 6 (t=15) A 0.284 0.303 0.288 0.344 0.294 0.290 B C 0.277 0.355 0.433 0.504 0.582 0.674 D E 0.662 0.396 0.483 0.635 0.685 1.131 F G 0.330 0.544 0.487 0.563 0.614 0.708 H 0.329 0.545 0.740 0.814 0.915 0.967 By 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 ALP, the cell lysate from practical 1 and the provided cell lysate. The activity of an enzyme can be measured by determining 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 initial 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 front 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 rates for each sample Sample Initial rate (Abs/min) Enzyme rate (mM/Min) Practical 1 lysate 0.1059 0.01059 Blank 0.0336 0.00336 Commercial ALP 0.0695 0.00695 Provided ALP 0.2745 0.02745 Discussion By 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 below: Cell lysate: (0.0059/0.00336) = 1.756 It 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) = 2.065 It 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) = 8.17 It can be said that the provided ALP sped up the reaction 8.17 times faster than without the provided ALP. Conclusion ALP 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 reaction 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.