1. Cell cultures and Cadmium treatment In this experiment, Arabidopsis thaliana is used. Its cell

600 Words3 Pages
1. Cell cultures and Cadmium treatment
In this experiment, Arabidopsis thaliana is used. Its cell suspension is cultured weekly by tenfold dilution. It contains 100ml fresh growth medium, 4.3g/L Murashige and Skoog basal salt mixture which is the mixture of ferrous sulfate heptahydrate, naphthaleneacetic acid, and kinetin. The salt mixture is then adjusted to its optimum pH. Cell cultures are incubated at 23C where the light and dark intensity are controlled. For this experiment, 16 hours of light and 8 hours of dark. Filter sterilized cadmium (Cd) chloride are used as its treatment. The concentration were manipulated from 0, 50, 100, 200, or 300 M. analysis of growth rate was done by taking out aliquots of 10mL at every several hours
…show more content…
Proteomic analysis, dark-grown and light-grown cells were treated with 200µM of CdCl2 or with equal volume of water as controls. Each treatment is replicated four times, which has 16 samples in total. The gels were imaged using Typhoon 9400 and analyzed by using DeCyder software. This software can generate the whole ratio of the gel. It uses the standardized spot abundance for statistical analysis. There will be spots of protein, but only ones that were treated with Cd was considered differently expressed protein.
4. Preparative gel electrophoresis and protein identification
350 of each sample are loaded on the gel. Based on Genomic Solutions, once fixing the changes of fresh 40% methanol and 10% glacial acetic acid for every 12 hour each, the gels were stained for 12 hours with Sypro Ruby solution in the dark. The gels were incubated in 10% methanol: 6% acetic acid for 4 hours for destaining procedure. The gels are the imaged by using ProPick Workstation. Triangulation and robotic excision are conducted for identification of protein spots. It was then submitted to tandem mass spectrometric analysis.
5. Measurement of lipid peroxidation and Cd content
Lipid peroxidation and cellular Cd content analysis were performed on treated cells for 72 hour with 200µM CdCl2. Cellular malondialdehyde was used as an indicator for lipid peroxidation. Calculation of amount of thiobarbituric acid reaction substance was done by determining the difference in optical density at

More about 1. Cell cultures and Cadmium treatment In this experiment, Arabidopsis thaliana is used. Its cell

Get Access