Protocol Isolation of proteins transfer of a tissue

Protocol: Isolation of proteins § transfer of a tissue sample into a tube § desintegration of the tissue by a lysis buffer containing SDS (sodium dodecylsultate) § separation of the lysate containing proteins from tissue fragments by centrifugation Determination of protein concentration § by the Bradford method § using BSA (bovine serum albumine) as a standard for calibration curve construction

Determination of protein concentration: § several methods are routinely used • the Bradford assay (we use in our experiment) • Lowry assay • BCA assay (Bicinchoninic assay) • ultraviolet absorbance assay, etc. § all of the listed methods rely on the use of a spectrophotometer (measurement of absorbance)

BSA 0. 1% 100 ugms per 100 ml 1 ugm per 1 ml 0. 1 ugms per 100 ul

Principle of the Bradford method § colorimetric assay based on absorbance shift of Bradford reagent that occurs after its binding to proteins § Bradford reagent contains Coomassie Brilliant Blue G-250 dye that binds to basic and aromatic amino acid residues (arginine (ARG), fenylalanin (PHE), tryptophan (TRY) a prolin (PRO)

Coomassie Brilliant Blue G-250 § when the dye binds to proteins, it is converted to blue color § the amount of this blue form is detected at 595 nm to quantify the concentration of proteins

Calibration curve

Bradford assay § preparation of standards for construction of a calibration curve – several samples with known concentration of protein (bovine serum albumine = BSA dissolved in water is routinely used) § dilution of our sample (lysate) with unknown concentration to fit into the concentration range of the calibration curve § incubation of standards and our samples with Bradford reagent § absorbance measurement (A 595) § construction of a calibration curve § determination of protein concentration in the lysate using the calibration curve

Another methods for protein determination: The Lowry assay § based on detection of tyrosine and tryptophan residues § blue color is developed and detectable with a spectrophotometer in the range of 500 -750 nm

Ultraviolet absorbance assay § determination of protein concentration by ultraviolet absorption (260 to 280 nm) § depends on the presence of aromatic amino acids in proteins (tyrosine and tryptophan) § [Protein] (mg/m. L) = 1. 55*A 280 - 0. 76*A 260

BCA method § BCA = bicinchoninic acid § the peptide bond itself is responsible for color development § purple color is detectable at 562 nm