Feruloyl Esterase Protocols - Revision history

Michael A. Speer: /* Method */

Michael A. Speer — Thu, 29 Mar 2012 17:50:38 GMT

Method

←Older revision		Revision as of 17:50, 29 March 2012
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	:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.		:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.
	:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.		:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.
-	:4. Put the control tube in the 99°C degree water bath for 3 minutes.<br>	+	:6. Put the control tube in the 99°C degree water bath for 3 minutes.<br>
-	:5. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>	+	:7. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>
-	:6. After two hours stop the reaction by 3 mins in 99 degree water bath.<br>	+	:8. After two hours stop the reaction by 3 mins in 99 degree water bath.<br>
-	:7. Measure absorbance in UV cuvette at 338nm.<br>	+	:9. Measure absorbance in UV cuvette at 338nm.<br>

	===Notes===		===Notes===

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Thu, 29 Mar 2012 17:50:18 GMT

Spectrophotometric Assay

←Older revision		Revision as of 17:50, 29 March 2012
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	===Method===		===Method===
-	~~The amount of substrate buffer is enough to do 5 samples (multiply accordingly).<br>~~
	:1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes		:1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes
	::*Three tubes for each culture to be assayed (label them)		::*Three tubes for each culture to be assayed (label them)

Michael A. Speer: /* Method */

Michael A. Speer — Thu, 29 Mar 2012 17:49:57 GMT

Method

←Older revision		Revision as of 17:49, 29 March 2012
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	::*For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).		::*For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).
	:2. Centrifuge 1 mL of each culture to be assayed.<br>		:2. Centrifuge 1 mL of each culture to be assayed.<br>
-	:3. Add 200ul of the appropriate culture supernatant to the 800 μL phosphate buffer.<br>	+	:3. Add 200ul of the appropriate culture supernatant to the tubes containing 800 μL phosphate buffer.<br>
	:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.		:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.
	:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.		:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Thu, 29 Mar 2012 17:49:26 GMT

Spectrophotometric Assay

←Older revision		Revision as of 17:49, 29 March 2012
Line 53:		Line 53:
	This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 338nm.		This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 338nm.
	===Materials===		===Materials===
-	*100mM sodium-phosphate buffer (pH = 7.3)	+	*100mM sodium-phosphate buffer (pH = 6.5)
	*Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)		*Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)
	*Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)		*Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Thu, 29 Mar 2012 17:48:42 GMT

Spectrophotometric Assay

←Older revision		Revision as of 17:48, 29 March 2012
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	===Notes===		===Notes===
-	*Don't forget to include a blank control (no enzyme)
-	*It's also a good idea to include a positive control (no substrate just ferulic acid)

	==References==		==References==

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Thu, 29 Mar 2012 17:48:25 GMT

Spectrophotometric Assay

←Older revision		Revision as of 17:48, 29 March 2012
Line 65:		Line 65:
	:2. Centrifuge 1 mL of each culture to be assayed.<br>		:2. Centrifuge 1 mL of each culture to be assayed.<br>
	:3. Add 200ul of the appropriate culture supernatant to the 800 μL phosphate buffer.<br>		:3. Add 200ul of the appropriate culture supernatant to the 800 μL phosphate buffer.<br>
		+	:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.
		+	:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.
	:4. Put the control tube in the 99°C degree water bath for 3 minutes.<br>		:4. Put the control tube in the 99°C degree water bath for 3 minutes.<br>
	:5. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>		:5. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>

Michael A. Speer: /* Method */

Michael A. Speer — Thu, 29 Mar 2012 17:46:25 GMT

Method

←Older revision		Revision as of 17:46, 29 March 2012
Line 60:		Line 60:
	===Method===		===Method===
	The amount of substrate buffer is enough to do 5 samples (multiply accordingly).<br>		The amount of substrate buffer is enough to do 5 samples (multiply accordingly).<br>
-	:1. ~~Make substrate~~ buffer ~~by combining:~~	+	:1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes
-	::*~~5ml 100mM sodium phosphate buffer~~	+	::*Three tubes for each culture to be assayed (label them)
-	::*~~15ul~~ ethyl-ferulate ~~solution~~ (~~10mg/ml)~~	+	::*For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).
-	~~:2. Make the~~ ferulic acid ~~blanks by combining the following:~~	+	:2. Centrifuge 1 mL of each culture to be assayed.<br>
-	~~::*5ml 100mM sodium phosphate buffer~~	+	:3. Add 200ul of the appropriate culture supernatant to the 800 μL phosphate buffer.<br>
-	~~::*15ul Ferulic Acid solution~~ (~~8.7mg/ml~~)	+	:4. Put the control tube in the 99°C degree water bath for 3 minutes.<br>
-	~~:3. Aliquot 1ml of these solution into 5 tubes for each solution~~.	+	:5. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>
-	:~~4. LABEL THE TUBES. <---don't forget this!!!<br>~~	+	:6. After two hours stop the reaction by 3 mins in 99 degree water bath.<br>
-	:5. Centrifuge ~~500ul~~ of each culture to be assayed.<br>	+	:7. Measure absorbance in UV cuvette at 338nm.<br>
-	:6. ~~For each culture, add~~ 200ul of supernatant to ~~each substrate solution~~.<br>	+
-	:~~:*This means every culture will have one ferulic acid tube, and one ethyl-ferulate~~ tube.	+
-	:5. ~~Incubate for 1 hour~~ in ~~37 degree~~ water bath.<br>	+
-	:6. ~~Stop~~ reaction by 3 mins in 99 degree water bath.<br>	+
-	:7. Measure absorbance in UV cuvette at ~~340nm~~.<br>	+

	===Notes===		===Notes===

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Thu, 29 Mar 2012 17:34:03 GMT

Spectrophotometric Assay

←Older revision		Revision as of 17:34, 29 March 2012
Line 51:		Line 51:
	===Notes===		===Notes===
	==Spectrophotometric Assay==		==Spectrophotometric Assay==
-	This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at ~~340nm~~.	+	This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 338nm.
	===Materials===		===Materials===
	*100mM sodium-phosphate buffer (pH = 7.3)		*100mM sodium-phosphate buffer (pH = 7.3)

Michael A. Speer: /* Spectrophotometric Assay */

Michael A. Speer — Sun, 11 Dec 2011 18:53:55 GMT

Spectrophotometric Assay

←Older revision		Revision as of 18:53, 11 December 2011
Line 53:		Line 53:
	This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 340nm.		This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 340nm.
	===Materials===		===Materials===
-	*~~50mM~~ sodium-phosphate buffer (~~use 0.135g monobasic and 0.217g dibasic to make 50ml of buffer~~ pH = 7)	+	*100mM sodium-phosphate buffer (pH = 7.3)
-	*Ethyl-ferulate solution (~~50mg~~/mL in dimethyl-formamide)	+	*Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)
		+	*Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)
		+	**To be a molar equivalent to the EF solution.

	===Method===		===Method===

Michael A. Speer: /* Method */

Michael A. Speer — Sun, 11 Dec 2011 18:52:41 GMT

Method

←Older revision		Revision as of 18:52, 11 December 2011
Line 57:		Line 57:

	===Method===		===Method===
-	The amount of substrate buffer is enough to do 10 samples (multiply accordingly).<br>	+	The amount of substrate buffer is enough to do 5 samples (multiply accordingly).<br>
	:1. Make substrate buffer by combining:		:1. Make substrate buffer by combining:
-	::*5ml ~~MOPS Buffer~~	+	::*5ml 100mM sodium phosphate buffer
-	::*~~3ul~~ ethyl-ferulate solution	+	::*15ul ethyl-ferulate solution (10mg/ml)
-	:2. ~~Centrifuge 500ul~~ of ~~culture~~.<br>	+	:2. Make the ferulic acid blanks by combining the following:
-	:3. ~~Add 100ul~~ of ~~supernatant~~ to ~~400ul of Sodium phosphate buffer~~.<br>	+	::*5ml 100mM sodium phosphate buffer
-	:4. ~~Add~~ 200ul of ~~buffered~~ supernatant to ~~400ul~~ substrate solution.<br>	+	::*15ul Ferulic Acid solution (8.7mg/ml)
-	:5. Incubate for ~~30 mins~~ in 37 degree water bath.<br>	+	:3. Aliquot 1ml of these solution into 5 tubes for each solution.
		+	:4. LABEL THE TUBES. <---don't forget this!!!<br>
		+	:5. Centrifuge 500ul of each culture to be assayed.<br>
		+	:6. For each culture, add 200ul of supernatant to each substrate solution.<br>
		+	::*This means every culture will have one ferulic acid tube, and one ethyl-ferulate tube.
		+	:5. Incubate for 1 hour in 37 degree water bath.<br>
	:6. Stop reaction by 3 mins in 99 degree water bath.<br>		:6. Stop reaction by 3 mins in 99 degree water bath.<br>
	:7. Measure absorbance in UV cuvette at 340nm.<br>		:7. Measure absorbance in UV cuvette at 340nm.<br>