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BIOCHEMISTRY LABORATORY Lab 3 Determination of Molecular Weight by SDS-PAGE Section: Friday March 10-03-2023 BY: Ekesie Jennifer 0400456
Abstract The experiment's goal was to use SDS-PAGE to determine the molecular weight of the unidentified proteins in samples A and B. The cassette is first filled with resolving gel (80%), set aside for polymerization, and then put in the electrophoresis unit. The remaining cassette space is then filled with stalking solution (80%), a comb is used to create wells, and the remaining cassette space is then filled and left to polymerize while the protein samples are being prepared. comparing the distances at which they migrated on an SDS-PAGE gel to those of reference proteins with known molecular weights. The gel's wells were filled with the protein samples, which were then electrophoretically separated and stained. The molecular weights of the proteins were estimated by graphing the log of their molecular weights against their migration distances, and the migration distances of the proteins were measured. The findings show that SDS-PAGE is an accurate technique for calculating the molecular weight of proteins. In conclusion, the experiment's goal was accomplished, and the outcomes were in line with the results attained. Results Reagent Amount Water 2.7 ml 4X resolving buffer 2 ml 30% acrylamide 3.2 ml 10% APS 80 ul Temed 3 ul Table 1 : resolving gel recipes for a 12% gel Pour the resolving gel solution into the gel cassette after thoroughly yet rapidly combining all of the ingredients. During pouring, it's crucial to watch out for air bubbles because they might disrupt the gel matrix and distort protein bands. Observe the gel's polymerization. You can load your protein samples for electrophoresis once the gel has set up. To get the correct protein load for each well given that your protein sample concentration is 4.65 L, you will need to dilute the sample properly with a sample buffer of 3.75ul. In general, ensuring consistent and repeatable outcomes in protein separation research requires adhering to a specific recipe for the resolving gel. You can get distinct and well-resolved protein bands by carefully adjusting the concentration of the reagents and protein samples. that makes it easier to determine a molecular weight accurately.
Specific details regarding lab 4 Figure 1: Gel image The top numbers show the well numbers after 70 minutes of electrophoresis. 10 mL of protein marker was put to well 4, 10 mL of given sample was applied to well 3. By measuring the distance that the proteins indicated by the bands travel, a standard curve is created utilizing the markers' distance and their known molecular weight to estimate the weight of the unidentified proteins. All measures in centimeters are taken from the top's horizontal line as the starting point. The distances between the bands the marker forms are calculated using the vertical line on the left as a reference. The proteins samples in well 2 and 3 produced the darkest band; the * indicates how far those proteins traveled. The 2 protein samples were produced, in a gel that contained 10% acrylamide. To further denature the proteins, these are subsequently heated. The electrophoresis lasted 70 minutes at a voltage of 250 V, a current of 66 mA. Coomassie blue was used to stain the gel plate, which was then left in the dye for some time before being rinsed with DI water. 2 1 3
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Distance migrated (cm) Molecular weight( KDa) Log of molecular weight 3.2 26 1.415 4.0 34 1.532 4.5 34 1.532 5.2 130 2.114 6.2 170 2.231 7.3 170 2.231 9.5 47.0 1.672 9.6 118 2.072 Table 2 summary of raw data for standard curve The proteins' migration was measured using SDS PAGE. The bands were compared using the EZ-Run Pre-stained Protein Marker to determine the molecular weight in kDa given in the lab manual, and their log was then computed to create the standard curve. Figure 2 distance migrated vs log of molecular weight
The protein migration distance is plotted in the figure versus the log of their molecular weight. The distance is shown on the x-axis, while the log values are shown on the y-axis. The graph's R2 value is 0.239, which is significantly lower than the recommended value of 0.97. This is typically brought on by insufficient electrophoresis time, insufficient resolving gel on the gel plate, or an error in reading the EZ-Run Prestained Protein Marker. The data shown here is taken from Table 2 and Figure 1 Observation sheet
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