20.109(S13):Purify protein (Day6) - Revision history

AgiStachowiak: /* For next time */

AgiStachowiak — Sat, 30 Mar 2013 17:40:37 GMT

For next time

←Older revision		Revision as of 17:40, 30 March 2013
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	==For next time==		==For next time==
-
-	~~<font color=red>Revise for S13</font color>~~

	#Calculate the approximate protein concentrations for your inverse pericams. First make a standard curve from the BSA data, then perform a linear fit (for example, using the ''Add Trendline'' function in Excel). The chart does not have to be especially pretty as it will not go in your report, but please do show your work, starting from the raw data. While for now mg/mL is an acceptable concentration unit, note that for your report you will want to convert to mM, μM, or nM - whichever seems most appropriate.		#Calculate the approximate protein concentrations for your inverse pericams. First make a standard curve from the BSA data, then perform a linear fit (for example, using the ''Add Trendline'' function in Excel). The chart does not have to be especially pretty as it will not go in your report, but please do show your work, starting from the raw data. While for now mg/mL is an acceptable concentration unit, note that for your report you will want to convert to mM, μM, or nM - whichever seems most appropriate.
-	#The vector [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf pRSET] has several properties that make it useful for protein expression and production in bacteria. Some of these were described in today’s Introduction, namely the T7 promoter and an ampicillin resistance gene. Name 1 other feature contained in the pRSET vector and tell what purpose it serves. (Use your own words to describe the purpose, don't just quote the catalogue.)
-	~~#Your Module 1 report revision is due by 11 AM next time, to the 20109.submit address and using a Firstinitial_Lastname_LabSection_Mod1-REV.doc naming scheme.~~
-	#Write a draft of the methods section (through today's experiments) to be included in your research article. Review the [[20.109%28S12%29:_Protein_engineering_report \| report-specific guidelines]] before you begin.
-	~~#Prepare a schematic that depicts your mutagenesis strategy and write a short caption for it. You might show proposed changes at both the nucleotide and amino acid level for E67K/T79P/M124S and X#Z.~~
	#The following analysis may not be included in your completed report, depending on whether your sequencing data was robust or not. We'll nevertheless take this opportunity to practice writing figures and results.		#The following analysis may not be included in your completed report, depending on whether your sequencing data was robust or not. We'll nevertheless take this opportunity to practice writing figures and results.
	#*Prepare a figure depicting the results of your diagnostic digest. Keep in mind the best practices for figures that we have discussed, from content to presentation. For example, the content should include the expected band sizes, and the presentation should include labeling of lanes as well as a few reference bands.		#*Prepare a figure depicting the results of your diagnostic digest. Keep in mind the best practices for figures that we have discussed, from content to presentation. For example, the content should include the expected band sizes, and the presentation should include labeling of lanes as well as a few reference bands.
	#*Write a few sentences of results section text to accompany your diagnostic digest figure.		#*Write a few sentences of results section text to accompany your diagnostic digest figure.
-	~~#Your second [[20.109%28S12%29:Reflection_assignments_summary_page \| reflection]] is due next time, on lessons learned from the report revision.~~
-	#[[20.109%28S12%29:Characterize_protein_expression_%28Day6%29 \| Day 6]] of this module will be an intense one, and has the potential to run long. You will do yourself a great service if you carefully read the text in advance, then consider what preparations you will need to do on that day, and organize your thoughts in your notebook and/or with your partner. This part will not be collected or evaluated with one exception. Fill in the table under "Advance preparation for PAGE" based on the OD values you measured. Be sure to post these to the [[Talk:20.109%28S12%29:Induce_protein_and_evaluate_DNA_%28Day5%29 \| Day 5 Talk page]] also if you haven't already.

	==Reagent List==		==Reagent List==

AgiStachowiak: /* Part 4: Protein Concentration */

AgiStachowiak — Sat, 30 Mar 2013 17:37:52 GMT

Part 4: Protein Concentration

←Older revision		Revision as of 17:37, 30 March 2013
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	===Part 4: Protein Concentration===		===Part 4: Protein Concentration===

-	#Prepare 12 mL Bradford reagent from the 5x concentrated stock by adding water.	+	#Prepare 12 mL Bradford reagent from the 5x concentrated stock by adding to a 9.6 mL water aliquot.
	#Obtain BSA standards from the teaching faculty.		#Obtain BSA standards from the teaching faculty.
	#*Each tube already contains exactly 10 μL of standard (or plain water, for your blank solution).		#*Each tube already contains exactly 10 μL of standard (or plain water, for your blank solution).

AgiStachowiak: /* Part 3B: Desalting */

AgiStachowiak — Sat, 30 Mar 2013 17:37:07 GMT

Part 3B: Desalting

←Older revision		Revision as of 17:37, 30 March 2013
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	#*The timing function on this centrifuge does not work! Bring your timer and manually turn the centrifuge off after 2 min. Start the timer once the rpm value approaches 2000.		#*The timing function on this centrifuge does not work! Bring your timer and manually turn the centrifuge off after 2 min. Start the timer once the rpm value approaches 2000.
	#*Because we all have to share one centrifuge, ideally spin with at least 2 other groups.		#*Because we all have to share one centrifuge, ideally spin with at least 2 other groups.
-	#When you remove your columns, the resin inside should be slanted~~, just as your polyacrylamide resin in Module 1 was~~. ~~Mark~~ right on the column where the highest point of the resin occurs, and orient that mark facing outward in the next step.	+	#When you remove your columns, the resin inside should be slanted. Make a mark right on the column where the highest point of the resin occurs, and orient that mark facing outward in the next step.
	#Transfer the column to a fresh 15 mL conical tube, and then gently apply your ~ 1 mL of protein to the center of the compacted resin.		#Transfer the column to a fresh 15 mL conical tube, and then gently apply your ~ 1 mL of protein to the center of the compacted resin.
	#Repeat the 2 min spin step just as before.		#Repeat the 2 min spin step just as before.

AgiStachowiak: /* Part 3A: Nickel-agarose purification */

AgiStachowiak — Sat, 30 Mar 2013 17:36:10 GMT

Part 3A: Nickel-agarose purification

←Older revision		Revision as of 17:36, 30 March 2013
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	#*Wash Buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9)		#*Wash Buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9)
	#*Elute Buffer (0.5 M NaCl, 10 mM Tris-HCl, 1 M imidazole, pH 7.9)		#*Elute Buffer (0.5 M NaCl, 10 mM Tris-HCl, 1 M imidazole, pH 7.9)
-	#*~~Each buffer contains~~ protease inhibitors to help keep your protein intact.	+	#*All but the Charge Buffer contain protease inhibitors to help keep your protein intact.
-	#Gently rock the ~~nickel-~~agarose resin to fully resuspend it, then distribute 400 μL of slurry to each of three tubes. Or, thank your TA if he has already pre-aliquoted them for you!	+	#Gently rock the agarose resin to fully resuspend it, then distribute 400 μL of slurry to each of three tubes. Or, thank your TA if he has already pre-aliquoted them for you!
-	~~#*The agarose beads in the resin were pre-charged with a nickel ion solution.~~	+	#Label each tube as wild-type or mutant, then spin for 1 min. at low speed.
-	# Label each tube as wild-type or mutant, then spin for 1 min. at low speed.	+
	#Remove the 200 μL of supernatant from the resin and add it to your waste collection tube. The damp, semi-solid resin left behind should be ~ 200 μL “tall.”		#Remove the 200 μL of supernatant from the resin and add it to your waste collection tube. The damp, semi-solid resin left behind should be ~ 200 μL “tall.”
	#First you must rinse the resin. Add 400 μL of sterile DI water to each tube. Place on the nutator for 15-60 seconds to mix, or simply invert the tube several times. Flick the tube to complete resuspension of the resin if necessary.		#First you must rinse the resin. Add 400 μL of sterile DI water to each tube. Place on the nutator for 15-60 seconds to mix, or simply invert the tube several times. Flick the tube to complete resuspension of the resin if necessary.
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	#Repeat steps 5 and 6 for the following buffers		#Repeat steps 5 and 6 for the following buffers
	#*a second wash with DI water		#*a second wash with DI water
		+	#*3 washes with Charge Buffer, 400 μL each time
	#*2 washes with Binding Buffer, 400 μL each time		#*2 washes with Binding Buffer, 400 μL each time
	#Add your entire cell extract to the resin (~550-600 μL, however much is left). Be sure to add each sample to the appropriately labeled tube!		#Add your entire cell extract to the resin (~550-600 μL, however much is left). Be sure to add each sample to the appropriately labeled tube!

AgiStachowiak: /* Protocols */

AgiStachowiak — Sat, 30 Mar 2013 17:33:13 GMT

Protocols

←Older revision		Revision as of 17:33, 30 March 2013
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	==Protocols==		==Protocols==
-
-	~~<font color=red>Now that this day is shorter, add stats?</font color>~~

	===Part 1: Lysis of cells producing wild-type and mutant IPC===		===Part 1: Lysis of cells producing wild-type and mutant IPC===

-	#You will be given an aliquot of room temperature BPER (bacterial protein extraction reagent), which also contains 0.1% bovine serum albumin (BSA, a stabilizer), and a protease inhibitor cocktail to guard against protein degradation. When you are ready to begin, add 1:1000 of cold lysing enzyme mixture (obtained from teaching staff) to the BPER solution.	+	#You will be given a 3 mL aliquot of room temperature BPER (bacterial protein extraction reagent), which also contains 0.1% bovine serum albumin (BSA, a stabilizer), and a protease inhibitor cocktail to guard against protein degradation. When you are ready to begin, add 1:1000 of cold lysing enzyme mixture (obtained from teaching staff) to the BPER solution.
	#Per cell pellet (6 total), add the appropriate volume of enzyme-containing BPER and resuspend by pipetting until the solution is relatively homogeneous.		#Per cell pellet (6 total), add the appropriate volume of enzyme-containing BPER and resuspend by pipetting until the solution is relatively homogeneous.
	#*Resuspend -IPTG samples in 300 μL, and +IPTG samples in 600 μL - do you remember why?		#*Resuspend -IPTG samples in 300 μL, and +IPTG samples in 600 μL - do you remember why?
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	#The following buffers have been prepared for you:		#The following buffers have been prepared for you:
		+	#*Charge Buffer (50 mM NiSO<sub>4</sub>)
	#*Binding Buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9)		#*Binding Buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9)
	#*Wash Buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9)		#*Wash Buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9)

AgiStachowiak: /* Introduction */

AgiStachowiak — Sat, 30 Mar 2013 17:31:36 GMT

Introduction

←Older revision		Revision as of 17:31, 30 March 2013
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	We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf (pRSET)] contains six Histidine codons downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these additional encoded residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.)		We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf (pRSET)] contains six Histidine codons downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these additional encoded residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.)

-	[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|~~right~~\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]	+	[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|center\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]
	<br style="clear:both;"/>		<br style="clear:both;"/>
	Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.		Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.

AgiStachowiak: /* Introduction */

AgiStachowiak — Sat, 30 Mar 2013 17:31:26 GMT

Introduction

←Older revision		Revision as of 17:31, 30 March 2013
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	[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|right\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]		[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|right\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]
		+	<br style="clear:both;"/>
	Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.		Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.

AgiStachowiak: /* Introduction */

AgiStachowiak — Sat, 30 Mar 2013 17:31:00 GMT

Introduction

←Older revision		Revision as of 17:31, 30 March 2013
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	Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in BL21(DE3) bacteria. Today you will isolate IPC from the bacteria, and you will begin characterizing your wild-type and mutant proteins.		Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in BL21(DE3) bacteria. Today you will isolate IPC from the bacteria, and you will begin characterizing your wild-type and mutant proteins.

-	We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf (pRSET)] contains six Histidine ~~residues~~ downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.)	+	We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf (pRSET)] contains six Histidine codons downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these additional encoded residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.)

	[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|right\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]		[[Image:20.109_Ni-Ag-Schematic.png\|thumb\|550px\|right\|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]]
-	Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while ~~irrelevant~~ proteins can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.	+	Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.

	[[Image:20.109_Histidine.png\|thumb\|80px\|right\|'''Histidine''']]		[[Image:20.109_Histidine.png\|thumb\|80px\|right\|'''Histidine''']]

AgiStachowiak: /* For next time */

AgiStachowiak — Thu, 28 Mar 2013 16:08:27 GMT

For next time

←Older revision		Revision as of 16:08, 28 March 2013
Line 175:		Line 175:

	#Calculate the approximate protein concentrations for your inverse pericams. First make a standard curve from the BSA data, then perform a linear fit (for example, using the ''Add Trendline'' function in Excel). The chart does not have to be especially pretty as it will not go in your report, but please do show your work, starting from the raw data. While for now mg/mL is an acceptable concentration unit, note that for your report you will want to convert to mM, μM, or nM - whichever seems most appropriate.		#Calculate the approximate protein concentrations for your inverse pericams. First make a standard curve from the BSA data, then perform a linear fit (for example, using the ''Add Trendline'' function in Excel). The chart does not have to be especially pretty as it will not go in your report, but please do show your work, starting from the raw data. While for now mg/mL is an acceptable concentration unit, note that for your report you will want to convert to mM, μM, or nM - whichever seems most appropriate.
-	#~~Remember~~ that by the ~~end~~ of ~~Day 7~~ you should ~~submit~~ your ~~final plans~~ for the ~~Module 3 experiment~~.	+	#The vector [http://tools.invitrogen.com/content/sfs/manuals/prset_man.pdf pRSET] has several properties that make it useful for protein expression and production in bacteria. Some of these were described in today’s Introduction, namely the T7 promoter and an ampicillin resistance gene. Name 1 other feature contained in the pRSET vector and tell what purpose it serves. (Use your own words to describe the purpose, don't just quote the catalogue.)
		+	#Your Module 1 report revision is due by 11 AM next time, to the 20109.submit address and using a Firstinitial_Lastname_LabSection_Mod1-REV.doc naming scheme.
		+	#Write a draft of the methods section (through today's experiments) to be included in your research article. Review the [[20.109%28S12%29:_Protein_engineering_report \| report-specific guidelines]] before you begin.
		+	#Prepare a schematic that depicts your mutagenesis strategy and write a short caption for it. You might show proposed changes at both the nucleotide and amino acid level for E67K/T79P/M124S and X#Z.
		+	#The following analysis may not be included in your completed report, depending on whether your sequencing data was robust or not. We'll nevertheless take this opportunity to practice writing figures and results.
		+	#*Prepare a figure depicting the results of your diagnostic digest. Keep in mind the best practices for figures that we have discussed, from content to presentation. For example, the content should include the expected band sizes, and the presentation should include labeling of lanes as well as a few reference bands.
		+	#*Write a few sentences of results section text to accompany your diagnostic digest figure.
		+	#Your second [[20.109%28S12%29:Reflection_assignments_summary_page \| reflection]] is due next time, on lessons learned from the report revision.
		+	#[[20.109%28S12%29:Characterize_protein_expression_%28Day6%29 \| Day 6]] of this module will be an intense one, and has the potential to run long. You will do yourself a great service if you carefully read the text in advance, then consider what preparations you will need to do on that day, and organize your thoughts in your notebook and/or with your partner. This part will not be collected or evaluated with one exception. Fill in the table under "Advance preparation for PAGE" based on the OD values you measured. Be sure to post these to the [[Talk:20.109%28S12%29:Induce_protein_and_evaluate_DNA_%28Day5%29 \| Day 5 Talk page]] also if you haven't already.

	==Reagent List==		==Reagent List==

AgiStachowiak: /* Protocols */

AgiStachowiak — Mon, 04 Feb 2013 02:46:18 GMT

Protocols

←Older revision		Revision as of 02:46, 4 February 2013
Line 21:		Line 21:

	==Protocols==		==Protocols==
		+
		+	<font color=red>Now that this day is shorter, add stats?</font color>

	===Part 1: Lysis of cells producing wild-type and mutant IPC===		===Part 1: Lysis of cells producing wild-type and mutant IPC===