SDS-PAGE Lab 5 PDF

BIOL 3120 - Cell Biology Lab Lab5. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) for Protein separation Rubaia Tasmin Master’s Student, Teaching Assistant 10/07/2024 Email: [email protected] 1  Overview of To...

BIOL 3120 - Cell Biology Lab Lab5. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) for Protein separation Rubaia Tasmin Master’s Student, Teaching Assistant 10/07/2024 Email: [email protected] 1  Overview of Today’s Lab 1. Introduction of protein gel electrophoresis  Types of protein gel electrophoresis  Components of polyacrylamide gel  SDS-PAGE 2. Lab Procedures 3. Lab 5 Report Requirements 2  Introduction of protein gel electrophoresis  Types of protein gel electrophoresis: - Native gel: separation is based on size, charge, and shape - Isoelectric focusing: separation is based on isoelectric point - SDS-PAGE: separation is based on size (Our focus in this lab!) - 2-dimensional gel: based on a two-step separation procedure - First-step: separation based on isoelectric point (Isoelectric focusing) - Second-step: separation based on size through SDS-PAGE 3  Components of polyacrylamide gel  Components of polyacrylamide gel:  Acrylamide: matrix material  N’N’-methylene-bis-acrylamide, known as “Bis”: cross-linking agent  TEMED: catalyst  Ammonium persulfate: initiator  The gel is made by the polymerization of acrylamide and bis, which forms a network of pores. Figure: Polymerization and crosslinking of acrylam 4  Components of polyacrylamide gel (cont.)  The pore size determines the rate of protein migration through the gel  The pore size is expressed as %T  The pore size decreases with increased %T  %T = total percentage of acrylamide (monomer) and bis- acrylamide (cross-linker) per 100 ml of solution. %T= Total monomer concentration %C= Weight percentage of crosslinker https://www.bio-rad.com/en-us/applications-technologies/introduction-polyacrylamide-g 5 els?ID=LUSPBRM5B  SDS-PAGE - Sodium dodecyl sulfate (SDS): anionic detergent that unfolds and denatures proteins, coating them in negative charge. - 2-mercaptoethanol: is a reducing agent that reduce disulfide bonds between proteins and unfold proteins to linear structure. - The proteins can now migrate in the same direction (from negative pole to Separation based on: positive pole) based on size. - Shape? - Charge? - Size 6  SDS-PAGE  Stacking gel: - Found at the top of the gel and contains the wells. - Has lower %T (larger pore size), lower pH. - All of the protein samples line up and enter the separating gel at exactly the same time.  Separating (a.k.a. resolving) gel: - Has higher %T (smaller pore size), higher pH. - Proteins will separate based on size.  Loading (a.k.a. dissociation) buffer: - Tris-HCl, SDS, glycerol, beta-mercaptoethanol, bromophenol blue.  Running buffer: - Tris, glycine, and SDS, pH 8.3. 7  Lab Procedures 1. Set the precast gel based on the video. https://www.youtube.com/watch?v=XnEdmk1Sqvg 2. Mix 5uL protein sample with 5uL loading buffer. 3. Heat the sample at 95oC-100oC for 5min, then load into the well. 4. Run the gel at 150V until the dye front reaches the bottom. 5. Stain the gel with 100mL Coomassie (CBB) stain solution. 6. Destain the gel with 100mL destain solution. 8 Relevant terms  Rf = Migration distance of protein from origin (bottom of well) Distance the dye front travelled from the origin Standard Curve= log10 of molecular weight (y-axis), Rf (x-axis) https://www.ruf.rice.edu/~bioslabs/studies/sds-page/rf.html 9 Post-lab Question Calculating the size of bands(arrows point 1.Fill the table for the marker and two Rf= band unknown bands. 2. Make the standard curve for the marker, migration and calculate Rf for two unknown bands. (cm)/dye front 3. Calculate these two unknown bands size. Molecular Retention Rf in Log10(mole (cm) wt of the factor (Rf) decimals cular ladder weight) 250 kDa 1/10 0.1 Log(25000 0) 150 kDa 100 kDa 75 kDa 50 kDa 35 kDa 25 kDa 10 kDa  Requirements for the Lab 5 Report 1. Introduction: Introduction of protein electrophoresis and principal of the SDS-PAGE. 2. Materials: All the chemicals used along with concentration, and instruments used. 3. Methods: Brief steps for the experiment you did. 4. Results: Analyzing the protein gel figure. 5. Discussion/conclusion: What improvements can you make? How can you justify the results? What is your conclusion? 6. Lab manual questions & underlined questions File name for your lab report submission- Last class slide BIOL3120_Section_Lastname_Firstname_ LabN​ # Send me the Word document by email: [email protected] 11 Group Lab Reports & Presentations Each group chooses one different topic from the following list:  Central Dogma of Molecular Biology  Spectrophotometry  Enzymes & Enzyme Kinetics  Gel Electrophoresis  DNA Extraction & Polymerase Chain Reaction Format:  Minimum of 3 pages (not including references)  Times New Roman, 12 font  Single spacing  1” margins Submission:  Each group submits one group report (10 points)  Submit by email: [email protected]  Report due date: Friday, November 8th  Presentation date: Tuesday, November 11th 12