Protocol: Isolation of proteins Determination of protein concentration 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 all of the listed methods rely on the use of a spectrophotometer (measurement of absorbance) the Bradford assay (we use in our experiment) Lowry assay BCA assay (Bicinchoninic assay) ultraviolet absorbance assay, etc.

BSA 0.1% 100 ugms per 100ml 1ugm per 1ml 0.1ugms 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 (A595) construction of a calibration curve determination of protein concentration in the lysate using the calibration curve

The Lowry assay Another methods for protein determination: 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/mL) = 1.55*A280 - 0.76*A260

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