16 I-BET151 Dialogue Tips
  • Concentrations of Gli or diclofenac in the above samples were analyzed by HPLC method developed in our laboratories. The separation was performed on a Waters SunFireTM C18 column (150?mm��4.6?mm, 5 ��m). The flow rate was kept constant at 1.0?mL/min and the temperature of oven was maintained at 40?��C. The mobile phase used in Gli separation was methanol �C0.2% acetic acid in water (63.5: 36.5, v/v). The mobile phase used in diclofenac detection is methanol�C0.1% acetic acid in water (70:30, v/v). The mobile phase was freshly prepared daily and filtered through 0.45?��m membrane filter. Gli and diclofenac were detected at 229?nm and 275?nm, respectively. The injection volume for all the samples was 20?��L. All concentrations were calculated from the standard curve of Gli and diclofenac obtained from spiked plasma samples. The methods were validated in terms of specificity, linearity and reproducibility. The result Oxygenase showed linearity of Gli and diclofenac over the concentration range of 0.0156�C1.5, 1.5�C15?��g/mL (r2=0.9994) and 0.469�C30?��g/mL I-BET151 purchase (r2=0.9996), respectively. The limit of quantification (S/N��3) of Gli and diclofenac in plasma was 3?��g/L and 8?��g/L. The limit of quantification (S/N��10) of Gli and diclofenac in plasma was 8?��g/L and 20?��g/L. The intra- and inter-day variations of Gli and diclofenac were less than 3%. Rats livers from normal rats and STZ-induced diabetic rats (n=3, each group) were immediately excised after the rats were sacrificed and total protein was isolated by RIPA lysis buffer. The supernatant fraction was obtained through centrifugation at 10,000��g for 10?min at 4?��C and then stored frozen at ?80?��C. Protein content was measured using the Micro BCA protein assay, using bovine serum albumin as standard protein. 100?��g protein was analyzed by a 10% SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF transfer membrane. Following the transfer, the membrane was blocked with 5% non-fat dried milk dissolved in the phosphate-buffered saline containing 0.05% tween 20 (TBST) for 1.5?h. The PVDF membrane was incubated with a 1/200��diluted GSK2656157 cell line rabbit anti-CYP 2C9 antibody in TBST containing 5% non-fat dried milk with shaking at room temperature for 2?h. After three washes with TBST, it was further incubated with peroxidase conjugated affinipure goat anti-rabbit IgG secondary antibodies, goat anti-rabbit IgG (diluted 1/5000 with TBST containing 5% non-fat dried milk) with shaking for 1.5?h and then washed three times with TBST. Hepatic CYP2C9 were detected by enhanced chemiluminescence on ?lm and quanti?ed by densitometry with a microcomputer imaging device. The ��-actin band was used as a loading control. The software of DAS 2.0 was used to estimate the pharmacokinetic parameters. Statistical analyses were performed using unpaired t-test. Numerical values in tables and figures were expressed as means��standard deviation (SD). Means were assumed to be statistically significant when P<0.05.</div>

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