User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06 - Revision history

Keyun Wang at 04:02, 8 December 2012

2012-12-08T04:02:27Z

Keyun Wang: /* Procedure for Protein Extraction and Purification from BL21(DE3) Cells */

2012-11-30T07:24:09Z

Procedure for Protein Extraction and Purification from BL21(DE3) Cells

←Older revision		Revision as of 07:24, 30 November 2012
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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 ~~______Sonicator~~.	+	** 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.

Keyun Wang: /* Procedure for Protein Extraction and Purification from BL21(DE3) Cells */

2012-11-29T05:52:33Z

Procedure for Protein Extraction and Purification from BL21(DE3) Cells

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T18:55:21Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 18:55, 25 November 2012
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	\|}		\|}
	* Based on the table, a graph of absorbance versus increasing mole ratios of Au/Lysozymes were made to better depict relationship between concentration of gold nanoparticles in supernatant and mole ratios of Au/Lysozymes. The graph is shown below:		* Based on the table, a graph of absorbance versus increasing mole ratios of Au/Lysozymes were made to better depict relationship between concentration of gold nanoparticles in supernatant and mole ratios of Au/Lysozymes. The graph is shown below:
		+	[[Image:KW.png\|350px]]
		+	* From the graph above, it can be shown that the concentration of gold nanoparticles in supernatant increases as the mole ratio of Au/Lysozyme goes from 20 to 40. The concentration of gold nanoparticles peaks at mole ratio of 40, then decreases from there on as ratio of Au/Lysozyme increases further. It seems like that the concentration of gold nanoparticles in supernatant increases from mole ratio of 120 to 130, but it might be caused by accident resuspension of fibers when absorbance of sample 130 Au/Lysozyme was taken.
		+	* The 40 mole ratio of Au/Lysozyme can be further investigated from 20 to 50 to determine an exact ratio of gold to lysozyme that encouraged protein aggregation.

	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T17:30:07Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 17:30, 25 November 2012
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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 indicated a peak at 530nm when gold nanoparticles ~~were formed~~. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below:	+	* 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 530nm 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'''

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T17:29:13Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 17:29, 25 November 2012
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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 Bakshi et al. which indicated a peak at 530nm when gold nanoparticles were formed. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below:	+	* The peak at wavelength 525nm was looked at separately based on [http://pubs.acs.org/doi/abs/10.1021/jp110296y Bakshi, et al], which indicated a peak at 530nm when gold nanoparticles were formed. A table listing absorbance at 530nm 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'''

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T17:28:00Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 17:28, 25 November 2012
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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 ~~530nm~~ was looked at separately based on Bakshi et al. which indicated a peak at 530nm when gold nanoparticles were formed. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below:	+	* The peak at wavelength 525nm was looked at separately based on Bakshi et al. which indicated a peak at 530nm when gold nanoparticles were formed. A table listing absorbance at 530nm 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'''
-	\| align="center" style="background:#f0f0f0;"\|'''Absorbance at ~~530nm~~'''	+	\| align="center" style="background:#f0f0f0;"\|'''Absorbance at 525nm'''
	\|-		\|-
	\| 20\|\|0.056		\| 20\|\|0.056

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T17:26:44Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 17:26, 25 November 2012
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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 ~~500nm~~ was looked at separately based on	+	* The peak at wavelength 530nm was looked at separately based on Bakshi et al. which indicated a peak at 530nm when gold nanoparticles were formed. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Au/Lysozyme ratio'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Absorbance at 530nm'''
		+	\|-
		+	\| 20\|\|0.056
		+	\|-
		+	\| 30\|\|0.077
		+	\|-
		+	\| 40\|\|0.269
		+	\|-
		+	\| 50\|\|0.235
		+	\|-
		+	\| 60\|\|0.193
		+	\|-
		+	\| 70\|\|0.036
		+	\|-
		+	\| 80\|\|0.036
		+	\|-
		+	\| 120\|\|0.048
		+	\|-
		+	\| 130\|\|0.083
		+	\|}
		+	* Based on the table, a graph of absorbance versus increasing mole ratios of Au/Lysozymes were made to better depict relationship between concentration of gold nanoparticles in supernatant and mole ratios of Au/Lysozymes. The graph is shown below:

	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Keyun Wang: /* Procedure for E.coli cell Transformation */

2012-11-25T17:12:19Z

Procedure for E.coli cell Transformation

←Older revision		Revision as of 17:12, 25 November 2012
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	* 40μL of E.coli cells was added into a 1mL microcentrifuge tube, 10μL of ADA E34K plasmid was added into bacteria without mix.		* 40μL of E.coli cells was added into a 1mL microcentrifuge tube, 10μL of ADA E34K plasmid was added into bacteria without mix.
	* The culture was placed on heat block for heat shock for 30 seconds. After heat shock, the culture was placed back on ice.		* The culture was placed on heat block for heat shock for 30 seconds. After heat shock, the culture was placed back on ice.
-	* 250μL of LB media was added to bacteria culture. The microcentrifuge tube was taped and attached to a small test tube. Sample was placed in orbital shaker under the following conditions:	+	* 250μL of SOC media obtained from New England Biolabs® was added to bacteria culture. The microcentrifuge tube was taped and attached to a small test tube. Sample was placed in orbital shaker under the following conditions:
	225rpm		225rpm
	37°C		37°C

Keyun Wang: /* Results for UV-vis on Au/Lysozyme samples */

2012-11-25T17:09:19Z

Results for UV-vis on Au/Lysozyme samples

←Older revision		Revision as of 17:09, 25 November 2012
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	[[Image:AuNP-Lysozyme_Absorbance.jpg\|600px]]		[[Image:AuNP-Lysozyme_Absorbance.jpg\|600px]]
	* 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.
	* The peak at wavelength 500nm was looked at separately based on		* The peak at wavelength 500nm was looked at separately based on

←Older revision		Revision as of 04:02, 8 December 2012
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	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
	==Purpose==		==Purpose==
-	* ~~Protein~~ was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography.	+	* 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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	** 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~~-vis spectrum~~ was taken at the eluted product over time and chromatogram was yielded. Because the chromatograph was not saved, the data cannot be displayed here.	+	** 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 ~~530nm~~ for each mole ratios of Au/Lysozyme is shown below:	+	* 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'''

←Older revision		Revision as of 05:52, 29 November 2012
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	** 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.
-	* The sample and water weight for balance were centrifuged in ~~_______instrument~~ with the following conditions:	+	* The sample and water weight for balance were centrifuged in Thermo Scientific® RC-6+ Centrifuge with the following conditions:
	Speed: 18,000rpm		Speed: 18,000rpm
	Temperature: 4°C		Temperature: 4°C
	Time elapsed: 2 hours		Time elapsed: 2 hours
-	Rotor used:~~_____~~	+	Rotor used: Sorvall F21S-8x50y
	* After centrifugation, supernatants and pellet form. The pellet was discarded, and about 100mL supernatant yielded and was kept in a 500mL beaker.		* After centrifugation, supernatants and pellet form. The pellet was discarded, and about 100mL supernatant yielded and was kept in a 500mL beaker.
	* The supernatant was filtered through Supor®-450 47mm membrane filter by pouring the supernatant through filter with membrane attached. Filter was attached to pressure suction that draws the supernatant through the membrane filter. The supernatant was filtered into a 800mL Erlenmeyer flask, then poured into a 25mL falcon tube.		* The supernatant was filtered through Supor®-450 47mm membrane filter by pouring the supernatant through filter with membrane attached. Filter was attached to pressure suction that draws the supernatant through the membrane filter. The supernatant was filtered into a 800mL Erlenmeyer flask, then poured into a 25mL falcon tube.
	* The filtered protein supernatant was injected into the GE Pharmacia® AKTA Purifier 100 Fast Protein Liquid Chromatography to separate ADA proteins from the rest of proteins in sample through the following procedure:		* The filtered protein supernatant was injected into the GE Pharmacia® AKTA Purifier 100 Fast Protein Liquid Chromatography to separate ADA proteins from the rest of proteins in sample through the following procedure:
-	** The column was ran using elution buffer with _____ at a speed of ~~___mL~~/s	+	** The column was ran using elution buffer with _____ 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 ~~_____~~	+	** 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_____~~	+	** Binding buffer was run through the column with _____at a speed of 5mL/min
	** UV-vis spectrum was taken at the eluted product over time and chromatogram was yielded. Because the chromatograph was not saved, the data cannot be displayed here.		** UV-vis spectrum 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.