Protein Structure & Purification PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected]  Learning outcomes  List the functions of proteins  List the Protein purification techniques  Explain Protein quantitation (Lowry and Bradford)  Define Proteomics Protein functions...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected]  Learning outcomes  List the functions of proteins  List the Protein purification techniques  Explain Protein quantitation (Lowry and Bradford)  Define Proteomics Protein functions Classification of proteins Protein purification  Protein purification  The basic aim in protein purification is to isolate one protein of interest from other contaminating proteins to study its structure and function, increasing its stability, and large-scale production. ▪ Proteins must be released from the cell to be purified. ▪ Purification of proteins is an essential first step in understanding their function. ▪ Purification techniques are based on the basic properties of proteins like solubility, size, charge, and binding affinity.  Ammonium sulfate precipitation  This technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations.  When low concentrations of salt is added to a protein solution the solubility increases.  In solution, proteins form hydrogen bonds with water molecules through their exposed polar and ionic groups. When high concentrations of small highly charged ions such as ammonium sulfate are added, these groups compete with the proteins to bind to the water molecules. This removes the water molecules from the protein and decreases its solubility resulting in precipitation.  Ammonium sulfate precipitation  The concentration of ammonium sulfate at which a protein comes out of solution and precipitates is different from other proteins in the mixture.  In general, higher molecular weight proteins will precipitate out at lower salt concentrations. This phenomenon of protein precipitation in the presence of excess salt is known as salting out. Salting in & out Salting in Salting out  Protein dialysis  Removal of salt molecules from the isolated protein solution through a semi permeable membrane.  The salt molecules move from the more concentrated solution to the less concentrated solution.  Based upon size of molecules.  Gel electrophoresis  Protein gel electrophoresis is a laboratory method used to separate mixtures of proteins according to molecular size.  Denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a discontinuous buffer system is the most widely used electrophoresis technique and separates proteins primarily by mass. ▪ The ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged.  Nondenaturing PAGE (native-PAGE), separates proteins according to their mass/charge ratio.  Protein purification  Will be explained in the upcoming chapters ▪ Column chromatography ▪ Ion exchange chromatography ▪ Affinity chromatography ▪ High Performance Liquid Chromatography (HPLC)  Proteins are purified from a mixture using combination of several techniques based on protein properties. Protein quantitation  Lowry protein assay  The Lowry protein assay (Folin–Ciocalteau method) is a biochemical assay for determining the total level of protein in a solution.  When the Folin reagent (a mixture of sodium tungstate, molybdate and phosphate), together with a copper sulphate solution, is mixed with a protein solution, molybdenum blue (blue color) is produced.  The amount of molybdenum blue can be quantified by its absorbance at λmax=750 nm.  Spectrophotometry  Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. ▪ The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. ▪ Every chemical compound absorbs, transmits, or reflects light over a certain range of wavelengths. Absorbance of two different compounds  Spectrophotometry  A spectrophotometer consists of two devices: ▪ A spectrometer: It produces a desired range of wavelength of light. ▪ A photometer: It detects the amount of photons that is absorbed by the chemical.  How to measure a sample? ▪ Switch ON the device. ▪ Blank measurement. Basic structure of spectrophotometers (illustrated by Heesung Shim) ▪ Sample measurement. ▪ Clean the device. ▪ Switch OFF the device.  Biuret test  Biuret test is a chemical test used to determine the presence of a peptide bond in a substance.  The method is based on the fact that proteins (and, as a rule, all substances containing two or more peptide bonds) react with copper to form a violet colored complex (absorption λmax= 454 nm), in the presence of excess copper, is proportional to the amount of protein present. Free AA and dipeptides do not undergo this test.  Bradford assay  Bradford assays are Coomassie dye-binding assays for fast and simple protein quantification. ▪ Samples are added to preformulated Coomassie blue G 250 assay reagent and the resultant blue color is measured at λmax= 595 nm following a short RT incubation.  The practical advantages of the method are: ▪ The reagent is simple to prepare. ▪ The color develops rapidly and is stable. ▪ Incubation at RT. Proteomics  The total DNA composition of a cell is referred to as the genome, and the study of the structure and function of this DNA is called genomics.  By analogy, the proteome is defined as the total protein component of a cell, and the study of the structure and function of these proteins is called proteomics. The aim of proteomics is to catalogue the identity and amount of every protein in a cell and determine the structure & function of each protein. References  Sanders ER. Aseptic laboratory techniques: plating methods. J Vis Exp. 2012;(63):e3064. Published 2012 May 11. doi:10.3791/3064