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Team:TU Delft/Project/solubility/characterization
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Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. ''Rajakumari et al'' (2006) Biochemical and Biophysical Methods | Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. ''Rajakumari et al'' (2006) Biochemical and Biophysical Methods | ||
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[[Image:TU_Delft_sudan_ii_balls_stick.png|thumb|right|350px|Ball-and-stick model of the Sudan II molecule, an orange-red azo dye used for staining of non-polar substances. Source [http://commons.wikimedia.org/wiki/File:Sudan-II-3D-balls.png Wikipedia]]] | [[Image:TU_Delft_sudan_ii_balls_stick.png|thumb|right|350px|Ball-and-stick model of the Sudan II molecule, an orange-red azo dye used for staining of non-polar substances. Source [http://commons.wikimedia.org/wiki/File:Sudan-II-3D-balls.png Wikipedia]]] | ||
Revision as of 09:19, 20 October 2010
Characterization of the Hydrocarbon Solubility Parts
Protein Production Analysis
The alna gene was induced when the culture reached a density of about 108 bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. Because the IPTG inducable protomor is generally regarded as verey strong, we expect that the resulting gel wil show when the expression of AlnA begins and when it reaches its maximum.
Emulsifier Assay
Example from literature Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. Rajakumari et al (2006) Biochemical and Biophysical Methods
That is why, instead of a colorless hydrocarbon, we choose to use the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves water. Meanwhile it has a nice absorbance peak at 493 nm, which makes it convenient to measure.
Calibration and Background
Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.
