User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06: Difference between revisions
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==Purpose== | ==Purpose== | ||
* | * ADA was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography. | ||
* Adenosine deaminase plasmid with the mutation E34K was transformed into DH5α-T1 E.coli cells with a second attempt. | * Adenosine deaminase plasmid with the mutation E34K was transformed into DH5α-T1 E.coli cells with a second attempt. | ||
* UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] to measure absorbance of supernatant of Au/Lysozyme samples | * UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] to measure absorbance of supernatant of Au/Lysozyme samples | ||
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* The frozen cells were placed into a 500mL beaker filled with ~30°C of warm tap water for 10 minutes. After 10 minutes, cells are completely thawed and appeared in liquid form. | * The frozen cells were placed into a 500mL beaker filled with ~30°C of warm tap water for 10 minutes. After 10 minutes, cells are completely thawed and appeared in liquid form. | ||
* On ice bath, the cells were sonicated in three 1 minute intervals with three repeated cycles of procedures below: | * On ice bath, the cells were sonicated in three 1 minute intervals with three repeated cycles of procedures below: | ||
** Cells are sonicated under 8mV for 30 seconds with | ** Cells are sonicated under 8mV for 30 seconds with Microson™ Sonicator 3000®. | ||
** Cells are immediately placed on ice for 30 seconds | ** Cells are immediately placed on ice for 30 seconds | ||
* After sonication, the E.coli cells were transferred from a 25mL falcon tube into a 30mL centrifuge tube. Another 30mL centrifuge tube was filled with tap water with the mass equal to the mass of 30mL centrifuge tube containing cellular sample. | * After sonication, the E.coli cells were transferred from a 25mL falcon tube into a 30mL centrifuge tube. Another 30mL centrifuge tube was filled with tap water with the mass equal to the mass of 30mL centrifuge tube containing cellular sample. | ||
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** Protein samples were injected into FPLC with a 10mL syringe and let run through the column at a speed of 5mL/min | ** Protein samples were injected into FPLC with a 10mL syringe and let run through the column at a speed of 5mL/min | ||
** Binding buffer was run through the column with _____at a speed of 5mL/min | ** Binding buffer was run through the column with _____at a speed of 5mL/min | ||
** UV | ** UV absorbance at 280nm was taken at the eluted product over time and chromatogram was yielded. Because the chromatograph was not saved, the data cannot be displayed here. | ||
** Fractions of protein was collected based on the UV-vis spectrum, and three separate fractions are formed as a result of affinity binding and competitive binding via imidazole. | ** Fractions of protein was collected based on the UV-vis spectrum, and three separate fractions are formed as a result of affinity binding and competitive binding via imidazole. | ||
** Fraction 1 and 3 from the column were collected and transferred into a 15mL falcon tube | ** Fraction 1 and 3 from the column were collected and transferred into a 15mL falcon tube | ||
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* From the graph above, it is shown that Au/Lysozyme with mole ratio of 40 yielded the greatest absorbance, and hence indicated the greatest number of gold nanoparticles in solution. Followed by 40 is mole ratio of 50, then mole ratio of 60 that yields decreasing concentration of gold nanoparticles in solution. This indicates that the concentration of gold nanoparticles decrease after the maximum mole ratio of 40. | * From the graph above, it is shown that Au/Lysozyme with mole ratio of 40 yielded the greatest absorbance, and hence indicated the greatest number of gold nanoparticles in solution. Followed by 40 is mole ratio of 50, then mole ratio of 60 that yields decreasing concentration of gold nanoparticles in solution. This indicates that the concentration of gold nanoparticles decrease after the maximum mole ratio of 40. | ||
* It was expected for mole ratio of 130 Au/Lysozyme sample to have an absorbance around 0.00. But according to graph, the absorbance for sample came out to be 0.04. This might caused by accidental resuspension of purple fibers into supernatant when withdrawing the supernatant for sampling. | * It was expected for mole ratio of 130 Au/Lysozyme sample to have an absorbance around 0.00. But according to graph, the absorbance for sample came out to be 0.04. This might caused by accidental resuspension of purple fibers into supernatant when withdrawing the supernatant for sampling. | ||
* The peak at wavelength 525nm was looked at separately based on [http://pubs.acs.org/doi/abs/10.1021/jp110296y Bakshi, et al], which stated that gold nanoparticles can be indicated through a peak at 530nm when gold nanoparticles are present in samples. A table listing absorbance at | * The peak at wavelength 525nm was looked at separately based on [http://pubs.acs.org/doi/abs/10.1021/jp110296y Bakshi, et al], which stated that gold nanoparticles can be indicated through a peak at 530nm when gold nanoparticles are present in samples. A table listing absorbance at 525nm for each mole ratios of Au/Lysozyme is shown below: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Au/Lysozyme ratio''' | | align="center" style="background:#f0f0f0;"|'''Au/Lysozyme ratio''' |
Revision as of 21:02, 7 December 2012
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Purpose
Procedure for Protein Extraction and Purification from BL21(DE3) Cells
Speed: 18,000rpm Temperature: 4°C Time elapsed: 2 hours Rotor used: Sorvall F21S-8x50y
Result for Protein Extraction and Purification from BL21(DE3) Cells
Procedure for E.coli cell Transformation
225rpm 37°C 1 hour
Following reagents were weighted out: 20g of LB Agar powder from Teknova® 5g of tryptone from Teknova® 2.5g of yeast extract from BD® 5g of NaCl from Fisher Scientific® 7.5g agar from BD® Above were placed into a 800mL Erlenmeyer flask. 500mL distilled water was added to reagents. The solution was autoclaved under liquid cycle (see 2012/11/03 for protocol for liquid cycle) After cooling to ~50°C, 50mg ampicillin was poured into autoclaved solution, swirled to mix Under sterile condition, the solution was poured into separate petri dish when temperature reaches ~40°C Plates were left under sterile condition to solidify
Results for E.coli cell Transformation
Procedure for UV-vis on Au/Lysozyme samples
20 - 30 - 40 - 50 - 60 - 70 - 80 - 120 - 130
Results for UV-vis on Au/Lysozyme samples
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