Electrophoresis Lab 8 PDF

Electrophoresis Couse: BT353 Semester: First 2024/2025 Teachers: Ghadeer Alsoukhni & Saif Alahmad Electrophoresis It is a method used to separate charged particles from one ot another based on differences in their migration speed. In the course of electrophoresis, two electrodes (typically...

Electrophoresis Couse: BT353 Semester: First 2024/2025 Teachers: Ghadeer Alsoukhni & Saif Alahmad Electrophoresis It is a method used to separate charged particles from one ot another based on differences in their migration speed. In the course of electrophoresis, two electrodes (typically made of an inert metal, e.g. platinum) are immersed in two separate buffer chambers. The two chambers are not fully isolated from each other. Electrophoresis Charged particles can migrate from one chamber to the other. By using an electric power supply, electric potential (E) is generated between the two electrodes. Due to the electric potential, electrons move by a wire between the two electrodes. Electrophoresis Electrons move from the anode to the cathode. Hence, the anode will be positively charged, while the cathode will be negatively charged. (Reduction) (Oxidation) Electrophoresis It is used to separate macromolecules based on: 1. Size 2. Charge 3. Shape Macromolecules Proteins, DNA, and RNA Electrophoresis Gel The gel has to achieve several general criteria to be applicable for biochemical electrophoresis. It needs to be: 1. Hydrophilic Ensures the gel can retain water, allowing it to form a stable matrix for electrophoresis. Supports the movement of charged biomolecules like proteins and nucleic acids. 2. Chemically stable The gel must not react with the biomolecules being analyzed or with the buffer solutions, ensuring reproducible and accurate results. 3. Neutral The gel itself should not carry a charge, which could interfere with the migration of biomolecules under the electric field. Electrophoresis Gel 4. Mechanically resistant Must withstand the physical stresses of handling, loading, and electrophoresis without tearing or deforming 5. With adjustable pore size Facilitates the separation of molecules of different sizes. For example: Polyacrylamide gels (PAGE) allow fine-tuning of pore size by adjusting the acrylamide and bis- acrylamide concentrations. Agarose gels are typically used for larger molecules like DNA, with pore sizes controlled by agarose concentration. Electrophoresis Gel It is always performed by using a special medium, most often a gel. Two compounds are dominantly used for gel electrophoresis: polyacrylamide and agarose. Polyacrylamide Polyacrylamide is a synthetic polymer widely used in biochemical and biotechnological applications, particularly for gel electrophoresis, due to its unique properties. Neurotoxicity: Acrylamide, a precursor of polyacrylamide, is neurotoxic and potentially carcinogenic. Handle with care, especially during gel preparation. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis): Used for protein separation based on molecular weight. Native PAGE: Separates proteins without denaturation, preserving their native state. DNA/RNA Gel Electrophoresis: Commonly used for high-resolution separation of nucleic acids. Agarose Agarose is another widely used gel matrix in electrophoresis, particularly for the separation of nucleic acids (DNA and RNA). It is derived from agar, a polysaccharide extracted from red algae. Nucleic Acid Separation Commonly used in gel electrophoresis to separate DNA or RNA based on size. The separation is facilitated by ethidium bromide (EtBr) or other dyes for visualization under UV light. Molecular Biology Techniques: PCR product analysis: Verifies the size and quality of amplified DNA. Restriction enzyme digestion analysis: Confirms successful cutting of DNA. Southern/Northern blotting: Used for transferring DNA or RNA to membranes. Electrophoresis Gel Agarose gels are used typically for the electrophoresis of large nucleic acids. Polyacrylamide gels typically provide much smaller pores than do agarose gels. The movement in electrophoresis occurs between: solid phase (gel) and liquid media (buffer) Electrophoresis Buffer The buffer in the medium serves two purposes: 1. One is to set and maintain the proper pH during electrophoresis. 2. The other function of the buffer is to act as conducting medium for the electric current. Agarose Gel Electrophoresis Agarose Gel Electrophoresis relies on the migration of charged molecules through an agarose matrix in an electric field. Nucleic acids are negatively charged due to their phosphate backbone and move towards the positively charged electrode (anode). The gel acts as a sieve, allowing smaller fragments to migrate faster than larger ones. Agarose gels are cast by dissolving the white agarose powder in an aqueous buffer. Agarose Gel Electrophoresis When the sample is heated to just below boiling, the agarose powder dissolves in the buffer to form a clear solution. The pore size of the gel can be controlled by the percentage of the agarose dissolved in the solution. A high percent agarose gel (say, 3% wt/v) will have a smaller pore size than a lower (0.8% wt/v) agarose gel. Key Principles of Agarose Gel Electrophoresis 1. Formation of a Porous Matrix: The network of agarose fibers creates pores, allowing nucleic acids to pass through. Concentration-dependent porosity: The size of the pores is inversely proportional to the agarose concentration. Low agarose concentration (0.5–1%): Larger pores, suitable for separating larger DNA fragments (1–20 kb). High agarose concentration (2–3%): Smaller pores, suitable for smaller fragments (50–500 bp). 2. Buffer Selection: Buffers like TAE (Tris-Acetate-EDTA) or TBE (Tris-Borate-EDTA) are used to maintain the pH and ionic strength during electrophoresis. Proper buffering prevents degradation of nucleic acids and ensures consistent migration patterns. Key Principles of Agarose Gel Electrophoresis 3. Thermal Stability: Agarose gels need to be melted and cooled to form a stable matrix. The gel solidifies as the agarose forms hydrogen bonds when cooled below ~40°C. 4. Incorporation of DNA Stains: Stains like Ethidium Bromide (EtBr) or safer alternatives (e.g., SYBR Safe, GelRed) can be added during gel preparation to bind nucleic acids and allow visualization under UV or blue light. 5. Well Formation: A comb is placed in the molten agarose to create wells where samples can be loaded. Proper well formation ensures consistent loading and separation.  Ethidium bromide Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. The standard concentration used in staining DNA in gels is 0.5- 1 ug/mL. Inserting itself between the base pairs in the double helix. EtBr is mutagenic, meaning it can cause mutations in DNA by intercalating between bases. It may also be carcinogenic, though its carcinogenicity in humans is not definitively proven. Agarose Gel Electrophoresis 100 bp -ve +ve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ladder Agarose Gel Electrophoresis (Materials) 1. Agarose gel preparation: Agarose powder Buffer (e.g., TAE or TBE) DNA stain (e.g., Ethidium) Comb and gel casting tray. Agarose Gel Electrophoresis (Materials) 2. Electrophoresis setup: Gel tank and power supply Loading dye (to visualize loading and monitor progress) DNA ladder/marker for size reference. Agarose Gel Electrophoresis (Materials) 3. Sample preparation: DNA/RNA samples Loading dye (e.g., glycerol or sucrose with tracking dyes like bromophenol blue). 4. Visualization: UV or blue light transilluminator. Agarose Gel Electrophoresis (Procedure) 1. Gel preparation: Dissolve agarose powder in the appropriate buffer by heating in the microwave. Cool the solution (~50°C), then add a DNA stain (Ethidium). Pour the gel into the casting tray with a comb in place to create wells. Allow the gel to solidify (~20-30 minutes). 2. Load the gel: Place the gel in the electrophoresis tank and add buffer to cover it. Mix DNA samples with loading dye. Load the samples and DNA ladder into the wells. 3. Electrophoresis: Connect the tank to the power supply and run the gel at a voltage (e.g., 80-120 V) until the dye front has migrated an appropriate distance. 4. Visualization: After electrophoresis, place the gel on a transilluminator. Observe and photograph the bands under UV or blue light. Agarose Gel Electrophoresis 100 bp -ve +ve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ladder SDS-PAGE SDS: sodium dodecyl sulfate. PAGE: polyacrylamide gel electrophoresis. SDS (Sodium Dodecyl Sulfate): A detergent that binds to proteins and denatures them, giving them a uniform negative charge. This eliminates differences in charge-to-mass ratios, allowing separation solely based on size. SDS binds at a ratio of ~1.4 grams of SDS per gram of protein. Polyacrylamide Gel: The gel acts as a molecular sieve. Smaller proteins move more easily through the gel matrix, while larger proteins are hindered, leading to size-based separation. The gel composition can be adjusted (e.g., varying acrylamide concentration) to optimize separation for specific protein size ranges. Polymerization process of SDS-PAGE gel The polymerization process of SDS-PAGE gel refers to the formation of the polyacrylamide gel matrix, which serves as the medium for electrophoretic protein separation. The process involves the polymerization of acrylamide and a cross-linking agent, typically N,N'- methylenebisacrylamide (BIS), to create a porous gel structure. Key Components of Polymerization: 1. Acrylamide: A monomer that forms the main structure of the gel. Its concentration determines the pore size of the gel (higher acrylamide = smaller pores). 2. BIS (N,N'-methylenebisacrylamide): A cross-linker that connects acrylamide chains, forming a three-dimensional network. 3. Ammonium Persulfate (APS): A chemical initiator that generates free radicals, driving the polymerization reaction. 4. TEMED (Tetramethylethylenediamine): A catalyst that accelerates the formation of free radicals from APS, ensuring rapid polymerization. Polymerization Reaction 1. Initiation: APS decomposes in the presence of TEMED to produce free radicals. These free radicals initiate the polymerization of acrylamide monomers 2. Propagation: Acrylamide monomers react with free radicals to form growing polymer chains 3. Cross-Linking: BIS reacts with acrylamide chains, forming cross-links that create the gel's three- dimensional network. Polymerization process Polymerization process Polymerization Reaction 1. Acrylamide/BIS Ratio: Adjusting this ratio changes the pore size of the gel. Typical concentrations: Low acrylamide (5–8%): Large pores for large proteins High acrylamide (12–15%): Small pores for small proteins. 2. pH: The polymerization reaction is pH-dependent, with the resolving gel (typically pH ~8.8) being more basic than the stacking gel (pH ~6.8). 1. Gelation Time: The amount of TEMED and APS controls the polymerization speed. Overuse of these reagents can lead to overly rapid gel formation and poor uniformity. Applications of SDS-PAGE 1. Protein Size Estimation: By comparing migration distances to a molecular weight marker (ladder). 2. Protein Purity Assessment: To check for contaminating proteins. 3. Post-Translational Modifications: Observing shifts in molecular weight due to modifications like phosphorylation. 4. Western Blotting: As a preparatory step before transferring proteins to a membrane. SDS-PAGE Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) systems are used to determine the size of protein chains or protein subunit chains in a protein preparation. Initially, the protein preparation is treated with an excess of soluble thiol (usually 2 mercaptoethanol) and SDS. The thiol reduces all disulfide bonds (–S–S–) present within and/or between peptide units. SDS-PAGE The SDS (an ionic or denaturing detergent) binds to all regions of the proteins and disrupts most noncovalent intermolecular and intramolecular protein interactions. These two components result in total denaturation of the proteins in the sample, yielding unfolded, highly anionic (negatively charged) polypeptidechains. SDS-PAGE SDS-PAGE SDS-PAGE Ladder KDa Procedure of SDS-PAGE 1. Preparing Gel Solutions: Running Gel (12-15% acrylamide, depending on the protein size): 3.75 mL of 30% acrylamide solution 3.75 mL of 1.5 M Tris-HCl (pH 6.8) 10 µL of 10% SDS 7.5 mL of distilled water 50 µL of 10% ammonium persulfate (APS) 5 µL of TEMED. Mix well and pour on top of the polymerized stacking gel. Insert a comb and let the gel polymerize for 30-45 minutes Procedure of SDS-PAGE 2. Preparing Protein Samples: Mix protein samples with 4X SDS sample buffer (containing SDS, glycerol, Tris-HCl, bromophenol blue, Beta-Mercaptoethanol or Dithiothreitol (DTT)). SDS: A detergent that denatures proteins by disrupting non-covalent bonds, causing them to unfold. Provides a negative charge to proteins, ensuring they migrate based on size during electrophoresis, not charge. Glycerol: Increases the density of the sample, ensuring it sinks to the bottom of the well when loaded onto the gel. Helps in protecting proteins during freezing. Tris-HCl (50 mM, pH 6.8-8.0): A buffer that maintains a stable pH during electrophoresis. Ensures that the sample remains at the proper pH for SDS denaturation. Bromophenol Blue: A tracking dye that allows you to monitor the progress of electrophoresis. Beta-Mercaptoethanol or Dithiothreitol (DTT): A reducing agent that breaks disulfide bonds within and between proteins, ensuring complete denaturation. Alternatively, DTT can be used for the same purpose, especially when beta- mercaptoethanol is not desirable due to its odor. Heat the mixture at 95-100°C for 5-10 minutes to denature the proteins and ensure they are fully coated with SDS. Procedure of SDS-PAGE 3. Electrophoresis Setup: After the gel has polymerized, remove the comb carefully and rinse the wells with running buffer (Tris-glycine-SDS buffer). Load the protein ladder into the first well (for size reference). Load the protein samples into the subsequent wells, ensuring they are properly added to avoid sample cross-contamination. 4. Electrophoresis: Place the gel into the electrophoresis tank and cover it with running buffer (Tris-Glycine- SDS). Connect the electrodes (anode to the bottom and cathode to the top). Run the gel at 80-100V for the stacking gel and increase to 120-150V for the resolving gel (until the dye front reaches the bottom of the gel). Procedure of SDS-PAGE 5. Staining and Visualization: After electrophoresis, the proteins can be visualized by staining. Coomassie Brilliant Blue Staining: Stain the gel in a Coomassie Brilliant Blue solution for 1 hour (gentle shaking). Rinse the gel with distilled water to remove excess stain. Destain the gel with a solution of 50% methanol and 10% acetic acid, changing the destaining solution every 30 minutes until clear bands appear.