2. 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

3. 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.

4. BSA 0.1%
100 ugms per 100ml
1ugm per 1ml
0.1ugms per 100 ul

5. 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)

6. 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

7. Calibration curve

10. 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

11. 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 nm

12. 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*A *A260

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