Biuret Protein Assay and Lab Assignment Protein Review What is the monomer of proteins Amino Acids Protein Review What is the basic structure of amino acids Amino group Carboxyl group Protein Review During polymerization of a peptide a condensation reaction results in what type of bond between each monomer Amide Group Formation When amino acids are joined by peptide bonds produces amide group Amide group CONH2 Protein Concentrations Biuret Protein Assay Biuret Reagent contains copper II sulfate light blue Amide groups complex with copper ions at a basic pH resulting in a violet blue color Biuret Benedicts Comparison Both contain copper and are light blue Benedict s Measures concentration of reducing sugars Involves redox reaction Color turns brick red Biuret Measures concentration of protein Involves complex formation not a redox reaction Color turns violet Biuret Benedicts Comparison Both result in a color change qualitative data Biuret Protein Assay Intensity of the color produced when exposed to peptides is Directly proportional to number of peptide bonds participating in the reaction therefore the amount of protein present Biuret Protein Assay How do we translate this to quantitative data Spectrophotometer Color changes can be measured by a spectrophotometer Quantitative measure of amount of light absorbed by solution When light passes through sample Some light is absorbed Some light is transmitted Spectrophotometer Each molecule has a wavelength of light that it absorbs Your reading represents absorbance abs of solution at a particular wavelength The Biuret complex absorbs light at 550 nm Where are proteins found in the cell Inside the cell Embedded within the membrane Extracellular matrix Protein Isolation Scientists often use detergent and mechanical disruption to isolate proteins for measurement Detergents such as SDS are amphipathic The detergents tend to form micelles when placed in aqueous solution Protocol Pick a Food Item to Determine Protein Concentration Estimate Protein Concentration mg g 1 oz 28 35 grams 1 ml 1 gram Convert to grams Convert to mg the divide by serving size Protocol Isolate your proteins Add biuret to form a copper complex Use a spectrophotometer to measure absorbance abs of light How does this give you the concentration of protein Biuret Protein Assay Standard Curve A standard curve is a type of graph used as a quantitative technique Multiple samples with known properties such as protein concentration are measured and graphed Biuret Protein Assay Standard Curve Samples with known properties are the standards We will be using BSA for our standards The graph you create from your BSA standards is your standard curve Biuret Protein Assay Standard Curve Standard curves allows the same properties such as protein concentration to be determined for unknown samples by using the slope of graph Biuret Protein Assay Standard Curve Example Unknown has an absorbance of 0 4 y Graph it out and estimate Use slope equation 65 ug ml 67 8 ug ml Determination of Protein Amount from Plant and Animal Sources Known Protein Concentration Tube 1 6 Set 1 Known Protein Concentration Tube 1 6 Set 2 Unknown Protein Concentration Will be using duplicates Known Protein bovine serum albumin BSA Unknown Proteins chicken fish egg white tofu milk chickpeas soy milk Procedure Creating Your Standards Known Protein Concentration How to Dilute Procedure Creating Your Standards The more BSA protein in the standard the higher the absorbance will be Procedure Creating Your Standards Construct STD CURVE graph to fill this out Must Calculate Clean Up Do NOT pour Biuret reagent down the sink Empty contents of test tube into waste container on instructor bench dry Thoroughly clean all test tubes and place upside down to Turn off spectrophotometer Empty food tube in the sink and rinse but use sieve to keep beads from sink Rinse beads and set aside to dry Place used BSA and Biuret tubes in container on instructor bench Wipe down bench top Straighten up bench top supplies