Micropatterning Proteins - Revision history

Admin: /* Patterning a coverslip. */

2024-07-02T16:03:04Z

Patterning a coverslip.

← Older revision		Revision as of 16:03, 2 July 2024
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	''Acid washed &/or plasma cleaned coverslips should be used for this procedure.''		''Acid washed &/or plasma cleaned coverslips should be used for this procedure.''

	# Dilute IgG~~<sup>1</sup>~~ to 1 mg/mL, preparing ~20 μL per coverslip to be patterned.		# Dilute IgG to 1 mg/mL, preparing ~20 μL per coverslip to be patterned<sup>1</sup>.
	# Tape a piece of parafilm to the bench.		# Tape a piece of parafilm to the bench.
	# Place a 20 μL drop of the IgG solution on the parafilm.		# Place a 20 μL drop of the IgG solution on the parafilm.
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	# Place the stamp on a cleaned coverslip and place a light weight (e.g. 1.5 mL tube filled with water) on top of the stamp.		# Place the stamp on a cleaned coverslip and place a light weight (e.g. 1.5 mL tube filled with water) on top of the stamp.
	# After 10-20 seconds, remove the stamp. If preparing other coverslips, repeat steps 3-8 for the additional coverslips.		# After 10-20 seconds, remove the stamp. If preparing other coverslips, repeat steps 3-8 for the additional coverslips.
	# Passivate the coverslip by placing 0.1 mg/Ml of poly(L-lysine)-graft-poly(ethylene glycol) [PLL-g-PEG] in PBS for 30 min at room temperature<sup>2</sup>.		# Passivate the coverslip by placing 0.1 mg/mL of poly(L-lysine)-graft-poly(ethylene glycol) [PLL-g-PEG] in PBS for 30 min at room temperature<sup>2</sup>.
	# Rinse once with water and store until needed.		# Rinse once with water and store until needed.
	# Clean the stamp and store, pattern-side-up, in a sealed container.		# Clean the stamp and store, pattern-side-up, in a sealed container.

Admin at 16:44, 19 June 2024

2024-06-19T16:44:11Z

← Older revision		Revision as of 16:44, 19 June 2024
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	<sup>2.</sup> Passivation prevents proteins and cells from adhering directly to the glass. Depending on the substrate, this step may or maynot be needed.		<sup>2.</sup> Passivation prevents proteins and cells from adhering directly to the glass. Depending on the substrate, this step may or maynot be needed.

			==== Citations ====

Admin: Created page with "This protocol is based on IgG micropatterning methods as published by Freeman ''et al''. Freeman et al., (2016) “Integrins Form an Expanding Diffusional Barrier That Coordinates Phagocytosis.” Cell. V164#1, pp 128-40. http://linkinghub.elsevier.com/retrieve/pii/S0092867415015676It may need to be optimized for other proteins. ==== Preparation of the Microprinting Stamp. ==== This is optimized for a PDMS based stamp and may have to be modified for stamps m..."

2024-06-19T16:43:38Z

Created page with "This protocol is based on IgG micropatterning methods as published by Freeman ''et al''. <ref>Freeman et al., (2016) “Integrins Form an Expanding Diffusional Barrier That Coordinates Phagocytosis.” Cell. V164#1, pp 128-40. http://linkinghub.elsevier.com/retrieve/pii/S0092867415015676</ref>It may need to be optimized for other proteins. ==== Preparation of the Microprinting Stamp. ==== This is optimized for a PDMS based stamp and may have to be modified for stamps m..."

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This protocol is based on IgG micropatterning methods as published by Freeman ''et al''. <ref>Freeman et al., (2016) “Integrins Form an Expanding Diffusional Barrier That Coordinates Phagocytosis.” Cell. V164#1, pp 128-40. http://linkinghub.elsevier.com/retrieve/pii/S0092867415015676</ref>It may need to be optimized for other proteins.

==== Preparation of the Microprinting Stamp. ====
This is optimized for a PDMS based stamp and may have to be modified for stamps made of different materials. It is best to perform this procedure after each use of the stamp; the stamp can then be briefly rinsed with 70% ethanol before use.

# Press the stamp gently against a piece of scotch take, and then pull the tape away.
# Soak stamp in diluted laboratory glassware cleaner, 20 min.
# Rinse 5 times with dH20.
# Rinse with 70% ethanol.
# Allow to air dry.

==== Patterning a coverslip. ====
''Acid washed &/or plasma cleaned coverslips should be used for this procedure.''

# Dilute IgG1 to 1 mg/mL, preparing ~20 μL per coverslip to be patterned.
# Tape a piece of parafilm to the bench.
# Place a 20 μL drop of the IgG solution on the parafilm.
# Place the stamp, pattern-side down, on top of the drop.
# Incubate at room temperature for 30 minutes.
# Lift the stamp and gently dry under a slow flow of air.
# Place the stamp on a cleaned coverslip and place a light weight (e.g. 1.5 mL tube filled with water) on top of the stamp.
# After 10-20 seconds, remove the stamp. If preparing other coverslips, repeat steps 3-8 for the additional coverslips.
# Passivate the coverslip by placing 0.1 mg/Ml of poly(L-lysine)-graft-poly(ethylene glycol) [PLL-g-PEG] in PBS for 30 min at room temperature2.
# Rinse once with water and store until needed.
# Clean the stamp and store, pattern-side-up, in a sealed container.

1. Unlabeled or fluorescently-labeled IgG can be used. If using unlabeled IgG, a fluorescent secondary antibody can be used to detect the IgG spots after the experiment is completed.

2. Passivation prevents proteins and cells from adhering directly to the glass. Depending on the substrate, this step may or maynot be needed.