Appendix E Lab Techniques

Appendix E: Lab Technique Lab Techniques Gel Electrophoresis Gel electrophoresis Separates macromolecules (proteins, DNA, or RNA). For proteins and small molecules the gel is polyacrylamide. For larger molecules (>500 bp), the gel is agarose. Negatively charged molecules travel toward the anode at the bottom. Large molecules will move SLOWER. Coomassie Blue stain can be used for visualization. − NEGATIVE cathode Native-PAGE: A polyacrylamide gel electrophoresis method for proteins using NON-DENATURING conditions. Proteins keep their native charge and structure so they are separated based on charge and size. SDS-PAGE A polyacrylamide gel electrophoresis method for proteins using DENATURING conditions. Sodium Dodecyl Sulfate denatures the proteins and gives the proteins a uniform charge. This allows them to be separated solely on mass , thus, you can estimate the protein’s molecular mass. Reducing SDS-Exactly the same as SDS-PAGE, but with the addition of a PAGE: reducing agent, b-mercaptoethanol, which will reduce the disulfide bridges and result in a completely denatured protein. Isoelectric A gel electrophoresis method that separates proteins on the Focusing: basis of their relative contents of acidic and basic residues. The gel has a pH gradient and the proteins will migrate through the gel until they reach the pH that matches their isoelectric point. At the pI, the protein has a neutral charge, so it will no longer be attracted to the anode and it will stop migrating. Southern Blotting: Detection of a specific DNA sequence in a sample. S D Northern Blotting: Detection of a specific RNA sequence in a sample. N R Western Blotting: Detection of a specific PROTEIN in a sample. W P Chromatography + POSITIVE anode Sanger DNA Sequencing Chain termination method. Uses dideoxy nucleotides. The ddNTP lacks a hydroxyl group on the 3’ carbon of the sugar ring. With the 3’ hydroxyl group missing, no more nucleotides can be added to the chain. The chain ends with the ddNTP, which is marked with a particular color of dye depending on the base that it carries. After mixing all components, it is virtually guaranteed that a ddNTP has incorporated at every single position of the target DNA strand. The strands are run through gel electrophoresis to separate them based on length. The colored dye is read and is used to establish the DNA sequence. Polymerase Chain Reaction Used to make many copies of a specific DNA region in vitro. The key ingredients of PCR are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized. Separates two or more molecules from a mixture. Primer: Must have high GC content and either a G or C at each end. Example: 5’-GCATAGAAGCATTCCGC-3’ Stationary Phase: Typically polar. Polar molecules elute slower. Mobile Phase: Typically nonpolar. Nonpolar molecules elute faster. Liquid Chromatography: Silica is used as the stationary phase while toluene or another nonpolar liquid is used as the mobile phase. Taq Polymerase: The DNA polymerase typically used in PCR. Named after the heat-tolerant bacterium from which it is isolated (Thermos aquaticus). Very heat-stable and most active around 70°C. Steps: 1. 2. 3. High-Performance Liquid HPLC is a type of liquid chromatography that uses high Chromatography: pressure to pass the solvent phase through a more finelyground stationary phase which increases the interactions between the moelcuels and the stationary phase. This gives HPLC higher resolving power. Denaturation (96°C) Annealing (55 - 65°C) Extension (72°C) Cycle is repeated until you have enough DNA Gas Chromatography: Vaporizes the liquid before separation. Molecules are separated based on polarity and boiling point. The stationary phase is a thin layer of material applied to the inside of the column. Typically the polarity of the stationary phase matches that of the solute. The mobile phase is an inert gas. Gel-Filtration Separates molecules by size rather than polarity. Smaller Chromatography: molecules enter the porous gel beads allowing them to (Size-exclusion) elute later. Larger molecules will elute faster because they do not fit in the pores and will not be slowed down. Ion Exchange Separates proteins by their net charge. The column is Chromatography: filled with charged beads, either POS or NEG. Cation Exchange: NEG beads used, NEG proteins elute 1st. Anion Exchange: POS beads used, POS proteins elute 1st. Affinity Chromatography: Separates proteins based on their affinity for a specific ligand. Beads are bound to a specific ligand and proteins with a high affinity for that ligand will bind to the beads. Proteins with a low affinity for the ligand will elute first. Thin-Layer Sheet coated in polar silica gel. Molecules are spotted on Chromatography: the bottom of the sheet. Sheet is placed in a nonpolar liquid. Mobile phase travels up the plate using capillary action. Nonpolar molecules have the highest Rf value. Thin-Layer Chromatography Solvent front Nonpolar Polar Retention Factor: 𝑅% =" '()*+,-.")/0*"102.' '()*+,-.")032.,*"%40,*"102.'