UGT1A4 protein levels were calculated against known titrated quantities of UGT1A standard, with quantification made relative to the levels of calnexin observed in each lane (quantified by densitometric analysis of western blots as already described).
The cells transfected with titrated quantities of the APP expression vector were subject to either thapsigargin treatment, to induce ER stress, or PBA, to stimulate AICD production, or left untreated as the control.
Disulfides (BSSB, GSSG) were prepared from titrated quantities of BSH and GSH, which were then oxidised by treatment with aqueous NH4HCO3 (33 m m) under aerobic conditions as previously described.
This study also pointed out the need of titrating the quantities of antibody and beads for each mark, as optimizing antibody to beads and antibody-coated beads to chromatin are not linear in reduction [ 7– 9], making these steps perhaps the greatest hurdle for small-scale ChIP.
Purified, non-fluorescent LapG possessing pAzF at positions V (top gel) or Y (bottom gel) was titrated into a fixed quantity of detergent solubilized LapD-sfGFP in the absence (left side of gel) or presence (right side of gel) of c-di-GMP (20 μM) and allowed to incubate for 20 min. Samples were irradiated with UV light for 5 min and analyzed by SDS-PAGE.
When measuring RNA quantity from HUVEC titrated minimal cell numbers, all of the isolated RNA was used in the Quantitect Reverse Transcription Kit (Qiagen).
The quantity of total RNA from titrated minimal cell numbers and vascular endothelial biopsies was measured following reverse transcription of the RNA to cDNA and subsequent real-time quantitative PCR along with a calibration curve of RNA from the same cells.
Therefore, some additional studies were performed in which MNP (100 nmol/mL) was progressively titrated by the repeated addition of initially limiting quantities of 90CE (20 nmol/mL) until the MNP was completely exhausted.
In lactic acidosis, for example, there is often a significant discrepancy between the blood lactate concentration and the base deficit and, more tellingly, when calculations are made during bicarbonate-based haemofiltration, it is apparent that significant quantities of acid other than lactic acid are being titrated by the administered bicarbonate.
However, it should be recognized that gender differences in GH responsiveness are largely eliminated nowadays when GH dose is titrated against serum IGF-I rather than being a fixed quantity based on body size.
Traditional qPCR methods were adapted for use with single cells to accurately quantify the fold differences between BCR-ABL and ABL expression levels down to less than single-cell quantities, as defined by typical quality control descriptions (such as linearity of the standard curve for titrated RNA, Figure S-1, Supporting Information).
