MD100 Medical Biochemistry I Lab Exercise 3 - SDS PAGE - Fall 2024 PDF

MD100 Medical Biochemistry I Lab Exercise 3: Introduction to SDS PAGE - Protein identification and characterization Fall Semester 2024  Objectives ▪ Introduction: SDS-PAGE laboratory technique Theoretical Background – Principle of SDS-PAGE ▪ Part A: Sample p...

MD100 Medical Biochemistry I Lab Exercise 3: Introduction to SDS PAGE - Protein identification and characterization Fall Semester 2024  Objectives ▪ Introduction: SDS-PAGE laboratory technique Theoretical Background – Principle of SDS-PAGE ▪ Part A: Sample preparation – dilutions ▪ Part B: Sample loading – Running the gel ▪ Part C: Gel staining and destaining ▪ Part D: Protein gel analysis – protein identification  Introduction to SDS-PAGE ▪ It is a technique used for the separation of proteins based on their molecular weight. ▪ The separation of macromolecules in an electric field is called electrophoresis. In SDS-PAGE, a discontinuous polyacrylamide gel is used as a support medium and sodium dodecyl sulfate (SDS) is used to denature the proteins. ▪ The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. ▪ SDS is an anionic detergent that denatures and binds to proteins to make them uniformly negatively charged. Therefore, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode. ▪ Reducing agents (e.g. β-mercaptoethanol) cleave the disulphide bonds of proteins and along with SDS disrupt the structure or protein. The principle of SDS-PAGE  The principle of SDS-PAGE High molecular weight proteins stay at the back while low molecular weight proteins migrate faster. They migrate from cathode(-) to anode side(+).  Protein identification Albumin Casein Molecular weight: 66.5 kDa Molecular weight: 24 kDa  Materials/Equipment Power Supplies Gels (Prepared in the lab or precast gels purchased from the market) Vertical Gel Electrophoresis Chamber Protein Samples Running Buffer (Tris/Glycine/SDS) Staining and Destaining Buffer Protein Ladder (Prestained protein molecular weight standards) Micropipettes and gel loading tips Laemmli Sample Buffer  Sample Preparation 10% UNKNOWN PROTEIN 5% 2,5% 1%  Sample Preparation Laemmli Sample Buffer preparation: PROTEIN DENATURATION 1.Tris-HCl pH 6.8. 2. SDS. 3. Glycerol. 4. 2-mercaptoethanol. 5. Bromophenol Blue. ▪ It is important to heat protein samples immediately after addition of Laemmli buffer, to prevent degradation of denatured proteins by some proteases which can be (partially) resistant to SDS- denaturation.  PROTOCOL 1. Prepare 1mL of the following dilutions using 10% unknown protein solution using water: ▪ 5% ▪ 2,5% ▪ 1% * Do not forget to label the tubes 2. Add 15μl of 5%, 2,5%, and 1% unknown sample to a new tube. (total 3 tubes – label them with your group name). 3. Add 5μl of Laemmli Sample Buffer (4x stock) into each sample(1/4 of total sample volume). Note: The loading buffer should be ¼ of the sample volume. Laemmli Sample Buffer contains 2-mercaptoethanol which can be toxic if ingested, and fatal if inhaled or absorbed through the skin.  PROTOCOL 4. Boil the samples to completely denature the proteins at 70°C (heat block) for 2 minutes exactly. Note: SDS breaks up the two- and three-dimensional structure of the proteins by adding negative charge to the amino acids. 5. Add freshly prepared 1 x Running Buffer to both chambers 6. Load 10μl of Protein Ladder at the first well of the gel. 7. Load samples (10µl) on electrophoretic apparatus. Note: The anode (+ electrode) must be connected to the bottom chamber and the cathode to the top chamber. The negatively charged proteins will move toward the anode. Gels usually run at a voltage that will run the tracking dye to the bottom as quickly as possible without overheating the gels. Overheating can distort the acrylamide or even crack the plates. The voltage to be used is determined empirically.  PROTOCOL 8. Run the gels at 180 volts for approximately 30 minutes. 9. When the dye front is nearly at the bottom of the gel, stop the run. Note: Before removing gels, the power must be turned off and cables removed. 10. Separate the plates and drop the gel into a staining dish containing deionized water. 11. Remove the water into waste and add Coomassie Blue staining solution for 15 minutes on a rocking table. Note: Add enough Coomassie Blue stain to cover the gel. 12. Pour off staining solution and rinse with deionized water to ensure the removal of Coomassie stain.  PROTOCOL 13. Add fresh Destain solution to cover the gel and incubate for 15 minutes on a rocking table, by changing the destain solution every 5 minutes (2 changes in total) 14. Remove the Destain solution and add deionized water overnight on a rocking table. 15. One of each group should come in the morning to take picture of the gel. Which is the protein in the unknown sample?  Protein gel analysis Lanes with one band indicate that the sample contains only one protein/protein subunit. Lanes with multiple bands indicate the presence of multiple proteins or multiple protein subunits/polypeptides. You can determine the identity of the proteins in each lane. Do the bands represent Albumin OR Casein? The relative darkness of the bands can imply the concentrations of the proteins in solution – you can estimate the protein concentration by the size of the band – if the concentrations are known. Which protein band represents the 5%, 2,5%, and 1% concentration of protein? Protein Ladder  Question 1 In an SDS-PAGE: A. Proteins are denatured by the SDS B. Proteins have the same charge-to-mass ratio C. Smaller proteins migrate more rapidly through the gel D. All the above  Question 2 Proteins can be visualized directly in gels by: A. Staining them with the dye B. Using electron microscope only C. Measuring their molecular weight D. None of these Results