SOLUTION: Electrophoresis Method for Protein Analysis | My Assignment Tutor

Page 1 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical ProcedureTITLE: Electrophoresis Method for Protein AnalysisSCOPE: Analysis of protein profiles from different sources usingSDS PAGE techniqueWRITTEN BY: Dr Aida Rajic DATE: 09. November 2020Hazards associated with the experiment:.).)Page 2 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical Procedure1 IntroductionElectrophoresis Method for Protein AnalysisThe purpose of this experiment is to compare protein profiles of tissues from saltwater fish (cod), fresh-water fish (trout) and chicken. In addition, we will beexamining the protein profile of value price fish-fingers, to try to determine thecomposition (i.e. predominantly made of cod or trout).We will separate the proteins based on their size and compare obtained proteinprofiles (see Figure 1, lanes 2-6)). Also, we will use the protein ladder (size marker,lane 1, Figure 1, and Figure 5), therefore the size of the proteins within our proteinprofiles could be elucidated.Figure 1: SDS-PAGE with different salt-water fish protein profiles obtained by our students. Byusing salt-water and fresh-water fish, and chicken, we should, hopefully, be able to obtain morevaried protein profiles.To this purpose, we are using Sodium Dodecyl Sulphate- Poly Acrylamide GelElectrophoresis (SDS-PAGE) method of protein analysis.SDS is a detergent that readily binds proteins. At pH 7, in the presence of 1%SDS and DTT (Dithiothreitol), proteins dissociate into their subunits and bindlarge quantities of SDS. Under these conditions most proteins bind about 1.4g1 2 3 4 5 6Page 3 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical Procedureof SDS per gram of protein. This completely masks the natural charge of theprotein giving a constant charge to mass ratio (Figure 2). Therefore, the largerthe molecule the greater the charge so the electrophoretic mobility of thecomplex depends on the size (mol. wt.) of the protein.Figure 2: SDS binds to a protein and in doing so produces a coat of negative charge around theprotein molecule. SDS-treated proteins can now be separated solely on the basis of their size.The sieving effect of the polyacrylamide is important in this technique and the rangeFigure 3: smaller protein molecules are able to move through the gel faster (further) than the largersized protein molecules.Page 4 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical ProcedurePolyacrylamide gels are formed by the polymerisation of acrylamide with a smallamount of crosslinking agent methylenebisacrylamide. However, mixing these twotogether will not form a gel. Polymerisation of the gel is initiated by addition ofpersulphate, usually in the form of ammonium persulphate, and TEMED(tetramethylethylenediamine) as the catalysts for the reaction (Figure 4). TEMEDaccelerates the rate of formation of free radicals from persulfate and these in turncatalyze polymerization. The persulfate free radicals convert acrylamide monomersto free radicals which react with inactive monomers to begin the polymerization chainreaction. The elongating polymer chains are randomly crosslinked bymethylenebisacrylamide, resulting in a gel with a characteristic porosity, whichdepends on the polymerization conditions and monomer concentrations.Figure 4: Polyacrylamide gels are formed by the polymerisation of acrylamide with a small amount ofcrosslinking agent methylenebisacrylamide. Addition of persulfate and TEMED initiates polymerisationThe pore size of the resulting polyacrylamide gel can thus be controlled by alteringthe concentrations of acrylamide and methylenebisacrylamide.Lower concentrations of acrylamide give gels with larger pores, which separate largemolecular weight proteins. In contrast, high concentrations of acrylamide give gelswith smaller pore size allowing better separation of small molecular weight proteins.Lower percentage gels are better for resolving very high molecular weight proteins,while much higher percentages are needed to resolve smaller proteins.The recommended concentrations of acrylamide are shown in Table 1 for thedifferent molecular weight ranges of protein molecules.Page 5 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical ProcedureTable 1: The recommended concentrations of acrylamide for the different molecular weight ranges ofprotein molecules.2 Materials, Equipment & Safety2.1. Reagents and Equipment2.1.1. Pre-prepared protein samples2.1.2. Tris Running Buffer: 0.375M TrisHCl, pH 8.8 + 0.2% SDS2.1.3. Coomassie dye2.1.4. Acetic acid for de-staining, 7%.2.1.5. Vertical Electrophoresis Tanks and power packs (30mA)2.1.6. SDS PAGE gel (10%)2.1.7. Fynn pipettes and tips, 10-100ul or 20-200ul2.1.8. Protein size marker, Thermo Scientific PageRuler Plus Prestained ProteinLadder2.2 Personal Protective Equipment2.2.1. Lab coat2.2.2. Gloves2.2.3. GogglesPage 6 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical Procedure3. Procedure3.1. Remove sample comb and bottom spacer from the electrophoresis plate.3.2. Place plates in the bottom reservoir and clip in place.3.3. Fill the top reservoir with running buffer filling the sample wells with no bubbles.Make sure there are no leaks from the top reservoir into the bottom reservoir.3.4. Fill the bottom reservoir with the running buffer3.5. Load 5 of protein size marker into the first well. Then loadyour samples in separate wells.3.6. Apply 30 mA current and leave to run. The run will take approximately 4 hours.You can stay for another 30 minutes/an hour to observe the movement of thetracking dye throughout the gel. At this point, you will stop with the experiment and Iwill take over to do the staining procedure. I will take photos of your gels and uploadthem on Brightspace for you to see. I will also keep the gels in the fridge (wrappedwell in parafilm to prevent them from drying out) so that you can look at them nexttime we are in the lab.3.7. When the tracking dye nearly reaches the bottom of the plate, turn off the powerand place the plate in a dish to be stained.3.8. Place Coomassie dye in the dish for 30 mins.3.9. Wash stain off with acetic acid.Page 7 of 7Ref No: SSPPIMDBIS107 – 02Faculty of Science Technology and HealthDivision of Science and TechnologyBiochemistry, IMDBIS107Students Standard Practical ProcedureFigure 5 Protein size marker (Thermo Scientific PageRuler Plus Prestained ProteinLadder)