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22.12.

2011

FATIH UNIVERSITY Genetics and Bioengineering Department Esra Nur EKMEKCOLU 07091022

Experiment 7: ANALYSIS of PROTEIN by SDS PAGE part: 2 Loading samples and Running the Gel Experiment Date: 15.12.2011 Section: A1

Lab Partners: Emine Mjgan ahin Mustafa Aksoy zge Ayta

Lab Instructors: Badeser Akdoan Umida Djakbarova

FALL 2011 - 2012

PURPOSE: The aim of this experiment is to learn how to analyze the proteins and separate them according to their molecular weight. INTRODUCTION: The separation of the molecules by using an electric field is called electrophoresis. 1 SDS Page (SDS Polyacrylamide gel electrophoresis) is the process that separates the proteins according to their molecular mass.2

Figure 1 : The SDS Page system. In the SDS Page process, proteins move from (-) to (+), or cathode to anode.

Figure 2 : Anode and cathode plates.

The cathode plate provides a positive charge, and the anode provides the negative charge. The difference in the voltage causes a power that is reason of the protein separation, and the reason of the proteins to move down the PAGE.3 The stacking gel is slightly acidic (pH 6.8) and has a low (5.5%) acrylamide concentration for making a porous gel. As an opposite, the separating gel is more basic (pH 8.8), and has a higher polyacrylamide content (12%), so the gel has narrower channels or pores.4 The protein that has a higher molecular mass is the slowest one, and the one that has the least molecular mass is the fastest. In SDS Page process, we use some chemical components. Loading Buffer: SDS DTT Glycerol Dye Buffer DTT: Its the reducing agent that reduces the bridges in proteins. Glycerol: If it doesnt exist, the proteins maybe not go down the wells, its used to make it easier for proteins to go down in the gel. Dye: It is used to see the rate of migration. The important point in this is, it doesnt interfere with the proteins or stain them. When the color reaches the bottom of the gel, the experiment is finished. Buffer: It maintains the pH.
Running Buffer: SDS Glycine Tris

Glycine: It is the source for electrons ( it gives electrons).

MATERIALS: Cassette Stacking Gel Separating Gel Loading Buffer (Fermentas Company) Running Buffer Comb Protein sample Loading Dye Molecular Weight marker (Fermantas/#26619) Falcon Tubes Micropipette

METHODS: The protein sample is taken from the instructors. 20l of this sample is put into both of the two falcon tubes. 5l loading buffer is added to the tubes. The tubes are labeled with marker. They are boiled at 100oC for 5 minutes. They are spinned at the max speed for 2 minutes. Previously prepared gels are used in the analyzing process. The comb is taken out gently. The wells are washed carefully with the running buffer. Running buffer is added to the upper and lower chambers. The samples are loaded to the wells in order. The gel is run at 90V, 25mA until the dye reaches the separating part. Then the voltage is changed to 220V, and its run until the dye is run out.

RESULT: The rate of the loading dye protein sample is calculated as follows: The appropriate Loading dye rate is 5x. It means that;

We used 20l protein sample, so the amount of loading dye must be 5l. (

DISCUSSION: In this experiment, we did the second and the last step of analyzing the proteins. We learned the preparation of gels before, but we used gels that arent prepared by us, because gels can only remain two days. The first thing we done is the procedure we did on the samples for making them suitable for this experiment. We put 20l protein sample to the falcon tubes. Then, we added 5l of the loading buffer in them. After that, we boiled this mixture at 100oC for 5 minutes. Time is really important in this process, because if we boil it more than 5 minutes the protein will break down. If we boil it less than 5 minutes, the protein will not completely denaturated. The next step is the centrifuge part. But this time, centrifuge isnt done for the supernatant. Supernatant doesnt occur at 1 minute. So we spinned our samples for just separating the proteins from each other, and get rid of the aggrigates. If they didnt separated before the analyzing part, they stick together and act like a single protein. As a result of this, they move very slowly. Then, we washed the wells with the running buffer. The reason of the foam in the running buffer is SDS (its a detergent). And we add loading buffer to the last well, loading buffer blocks the horizontal movement of the proteins (especially in the stacking gel). The optimum protein amount for this experiment is 20 g 60 g. If its too low, we cant observe, if its too much we cant see a proper bonding. After the cassette and the gels were ready, we started to load the gels in order. We prepared two samples, so everyone can load the gels and everyone in the class experienced in this process. There are some points for a proper loading: A careful pipetting: While we are taking the sample with a pipette, we have to avoid air bubbles, and if there are air bubbles occurred in the pipette, we have to get rid of them before loading. The angle of the pipette: We have to hold the pipette in a vertical position. Pulling the pipette out: We mustnt raise our finger from the top of the pipette before. This is maybe the most important of them all because, if we raise our hand, the pipette will take the gel out, and corrupts it

As a result, this experiment thought us how to analyze the proteins by the SDS Page method, load the samples properly, and minimize the mistakes done in the experiments.

REFERENCES: 1) Experimental Biosciences http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html 2) School of Life Sciences http://askabiologist.asu.edu/sds-page 3) Penn State Julie Simmons http://www.personal.psu.edu/jms5704/blogs/simmons/technical-definition.html 4) Smith College http://www.science.smith.edu/departments/Biochem/Biochem_353/sdspage.html

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