Isolation, Protein Analysis and Subcellular Fractionation of Mitochondria
Introduction
Several paragraphs describing the background to the different parts of the experiment and how they support the main focus of the experiment (subcellular fractionation of mitochondria by centrifugation, why use SDH etc?).
Aim
The aim of the experiment is to isolate mitochondria from toad liver, analyze subcellular fractionation of mitochondria by centrifugation and analyze protein using BCA assay.
Methods
The toad liver was prepared by technical staff. The whole cell lysate (WCL) was pelleted and the supernatant was obtained and placed in the MITO (mitochondrial fraction) tube. Next it was centrifuged three times to obtain three supernatant solutions labelled SN1, SN2 and SN3. Isolation-buffer was used to resuspend the pellet and to isolate subcellular fractions from the cell lysate by differential centrifugation. Next a BCA assay was used to determine the protein concentration of the SN1, SN2, SN3, WCL and MITO tubes using absorbance of 595nm and a BCA standard curve was obtained. Mitochondria was isolated by testing for the presence of succinate dehydrogenase activity. It analyzed subcellular fractions for evidence of mitochondrial enzyme. Don't use plagiarised sources.Get your custom essay just from $11/page
Results
| Table A: Concentration of Reduced INT in Sample | ||
| Sample | Absorbance 490nm | Concentration of reduced INT (umol/L) |
| WCL | 0.255 | 10.1634 |
| SN1 | 0.437 | 17.4172 |
| SN2 | 0.012 | 0.4783 |
| MITO | 0.362 | 144.281 |
| Sample | Absorbance 595nm | Concentration of Protein (ug/ml) | Total Amount of Protein (ug) |
| WCL | 1.881 | ||
| SN1 | 1.471 | ||
| SN2 | 0.533 | ||
| SN3 | 0.246 | ||
| MITO | 0.374 |
In this section, you should state your findings in writing. You will need at least two paragraphs of text. Include your tables and figures. Make them look professional. Indicate units and provide titles, legends and numbers for figures. Tabulated results and figures must be referred to in the text of the Results section. Include all calculations in the report or as appendices.
Discussion
This section should address the significance or meaning of the results. You can use subheadings for each section if you like. Mention errors and complications that may have occurred. Discuss the significance or likely effect of errors on your results and how you interpret the data. Compare your observations to similar experiments in published in journal articles. How do your results relate to expected or previously obtained results for similar experiments? Use in-text citations when you refer to the research of others and include a reference list at the end. You should include a conclusion to your experiment in this section, linking your findings to your aims, and you should also include suggestions for improvement or further research.
References