4 - Lecture Guide

Review of Lecture 4 Questions to think about…. Why is protein purification necessary? ALLOWS FOR THE TESTING AMINO ACIDS AND THEIR ANALYSE TO FIND THE SEQUENCE. It helps understand the structure, function, and specific activity of the protein, in addition to helping us understand the relationship between the structure and its function. Why should protein purification be carried out at low temperatures? TO PREVENT DENATURATION AT 0 TO 4 DEGREES CELCIUS. Which protein purification and separation techniques separate proteins on the basis of size? SIZE-EXCLUSION CHROMATOGRAPHY What technique might you use to separate two proteins that co-migrate on an SDS polyacrylamide gel? 2D-ELECTROPHORESIS Which technique separates proteins on the basis of charge? ION exhange chromatography ION-EXCHANGE CHROMATOGRAPHY… CATION EXCHNAGE WHERE CELULOSE OR AGAROSE BEADS ARE BOUND TO BY NEGATIVE ‘DEAE’ COMPOUND. ANION EXCHANGE IS WHERE CELLULOSE OR AGAROSE BEADS ARE BOUND TO BY POSITIVE COMPOUNDS. Which techniques separate proteins on the basis of binding specificity? Affinity Chromatraphy What does a single band on a protein gel represent? THIS REPRESENTS A SINGLE SUBUNIT. THIS CAN MEAN DIFFERENT THINGS IN DIFFERENT STUDIES. IN ISOELECTRIC FOCUSING THIS COULD PRESENT THAT MOLECULARE WEIGHT OF A SINGLE PROTEIN AND ITS ‘PI’ POINT, WHERE IT CARRIES NO CHARGE. What is SDS, and why is it used in electrophoretic procedures? SODIUM DODECYL SULPHATE IS USED AS A DETERGENT IN BREAKING NONCOVALENT BONDS FOUND IN SECONDARY AND TERTIARY STRUCUTRE. THIS IS NOT BE CONFUSED WITH MERCAPTOETHANOL OR DTT (DITHIOTHREITOL) Explain why two-dimensional gel electrophoresis is a more sensitive analytical method than one-dimensional gel electrophoresis? Why is it important to be able to measure the amount of a specific protein in a sample? This tells us the purity of the protein isolated from the cell and its SPECIFIC ACTIVITY. The higher the specific activity, THE MORE PURE A SAMPLE IS AND THIS NUMBER INCREASES WITH MORE PROTEIN PURIFICATION TECHNIQUES USED. Describe ion-exchange chromatography. What are the two types? CATION EXCHANGE IS WHERE BEADS ARE NEGATIVELY CHARGED SO THAT THEY ATTRACT POSTIVELY CHARGED CATION. THE OPPOSITE IS TRUE FOR ANION EXCHANGE. What are 2-mercaptoethanol and dithiothreitol used for? REDUCING AGENTS What is “salting out”? THIS IS THE PROCEDURE AFTER THE CRUDE SAMPLE OF PROTEIN HAS BEEN FRACTIONATED (THAT IS, AFTER THE CELLS HAVE BEEN BROKEN BY EITHER FRENCH PRESS, DETERGENTS, SONIFICATION, HOMOGENIZERS, MECHANICAL STRESS (BLENDERS)) AND AFTER THE CRUDE SAMPLE HAS BEEN CENTRIFUGED (500, 10000, 100000 G OF CENTRIFIGUATION AT 10, 20, AND 60 MINUTES RESPECTIVELY, YEIELDING NUCLEAR FRACTIONS, MITROCHONDRIAL FRACTIONS, AND MICROSOMAL FRACTIONS, RESPECTIVELY). Salting out refers to the process where you add a particular type of salt, such as ammonium sulphate at high concentrations to a crude extract. Generally, the higher the salt concentration, the lower a proteins solubility in a cellular extract. The proteins are no longer soluble so they precipitate. Describe how a Coomassie Blue stained protein gel can be used to estimate the molecular weight of a protein. THE COOMASSIE BLUE STAINED GEL CAN BE PUT UNDER ULTRA VIOLET LIGHT TO SEE HOW FAR THE BANDS OF PROTIENS HAVE TRAVELLED IN COMPARISON TO A BAND OF KNOWN PROTEINS. What is MALDI-TOF? Why is this a useful technique? USED IN ACCORDANCE TO MASS SPECTOMETRY FOR PROTEINS THAT ARE USUALLY FRAGILE or SENSITIVE TO IONIZATION used in REGULAR TECHNIQUES. THE PROTEIN IS EXPOSED TO A LASER LIGH THAT IONIZES THE PROTEIN ONTO A MEDIUM.. THE PROTEIN IS THEN VACUUMED AND SENT TO A DETECTOR SUCH AS A MASS SPECTOMETRY UNIT. What is ESI-MS? Why is this a useful technique? ELECTROSTATIC IONIZATION IS A TECHNIQUE WERE PROTEINS ARE FORCED INTO A GAS PHASE BY HAVING THEM PASS THROUGH A HIGH VOLTAGE ELECTRODSTATIC NEEDLE. THE SAMPLE IS THEN SPRAYED AS A FINE MIST AND DETECTED VIA MASS SPECTOMETRY. Why is dialysis of protein important for a purification scheme? THIS PROCEDURE USUALLY OCCURS AFTER THE PROCESS OF SALTING OUT IN ORDER TO SEPARATE THE SALTED PROTEIN WHERE THE SALT MOVES OUT OF THE DIALYSIS TUBE AND THE PROTEIN CONTENT (WHICH IS TOO LARGE TO MOVE OUT) IS SEQUENTERED IN THE DIALYSIS TUBING. Be familiar with the different types of chromatography discussed in class. Column chromatography: ion exchange, size exclusion, affinity, high pressure liquid chromatography. What is an anode? A cathode? ANODE IS THE POSITIVE END, CATHODE IS THE NEGATIVE END WHERE POSITIVE CATIONS ARE ATTRACTED TO.