CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

36
2.11.1 Preparation of total cell lysis protein
A549 cells (2.0 x 10 5 cells) were seeded in each 6 well plates, and exposed to a
test sample of various concentrations at 37 2 incubator for 24 h. The
cells were collected and the pellet was lysed with 200 µl of ice-cold lysis buffer (10
mM Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% vol/vol
-2-mercaptoethanol) and incubated on ice for 30 minutes. The
pellet was then centrifuged at 12,000 x g at 4 was
quantified for protein using a Bio-Rad Protein Assay (Bio-Rad).
2.11.2 Protein determination
The protein concentration of cell pellets was quantified by the Bio-Rad Protein
Assay (Bio-Rad). When Coomassie dye in the protein assay kit binds to proteins in
acidic medium, there is an immediate shift in absorption maximum occurs from 465
nm to 595 nm with a concomitant color change from brown to blue. Protein
concentrations are estimated by reference to absorbance obtained for a series of
standard protein dilutions, which are assayed alongside the unknown samples.
as follows:
The procedure of protein determination by Bio-Rad protein assay reagent is
1. Bovine serum albumin (BSA) standard solution in various concentrations
(187.5 – 500 µg/ml) was prepared from stock BSA (2,000 µg/ml).
2. Pipette each standard and sample into the appropriate microplate wells.
3. Add 250 µl of Bradford reagent to each well and mix.
4. Measure the absorbance at 620 nm with a microplate reader.