CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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35 min, followed by the amplification process, which are denaturation at 95 C for 30 sec, annealing at optimal temperature of each gene for 30 sec and extension at 72 C for 40 sec, and the final extension step at 72 C for 5 min. The optimized conditions resulted in the efficient amplification of DNA without non-specific amplified products. 2.10.5 Agarose gel electrophoresis and analysis of PCR product After amplification, 10 µl from each sample was electrophoretically separated on a 2 % (wt/vol) agarose gel and stained with ethidium bromide (EtBr, Vivantis). The DNA bands were visualized by UV illumination and documented by using a Bio- Rad gel doc 1000 system. The intensity of band was quantified by Quantity One Software (Bio-Rad). The standard 100-base pairs DNA ladder was used as molecular weight marker. The results are expressed as the percentage of gene expression. The expression was calculated by using the formulae below: % Gene expression = Mean band intensity of interested gene x 100 2.11 Western blot analysis Mean band intensity of GAPDH The Western blot analysis is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or cell extract. It uses gel electrophoresis to separate denatured proteins by the size of the polypeptides. The proteins are then transferred to a membrane, where they are probed using an antibody specific to the target protein.
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.
Page 1 and 2: 2.1 Chemicals and materials CHAPTER
Page 3 and 4: Name 24 Table 2.2: Sequences of oli
Page 5 and 6: 2.6 DNA polymerase stop assay 26 DN
Page 7 and 8: 2.9 Cytotoxicity test 28 Cell survi
Page 9 and 10: 30 with 5% CO2. The first plate, as
Page 11 and 12: 32 Table 2.3 Primer sequences for d
Page 13: 34 2.10.3 cDNA synthesis by reverse
Page 17 and 18: 38 through polyacrylamide gel. Smal

CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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