Isolating extracellular protein from Streptomyces - Revision history

Sam Prudence: /* Isolation of extracellular protein from Streptomyces */

2019-09-12T16:02:37Z

Isolation of extracellular protein from Streptomyces

← Older revision		Revision as of 16:02, 12 September 2019
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	=Isolation of extracellular protein from ''Streptomyces''=		=Isolation of extracellular protein from ''Streptomyces''=
	This protocol can be used to isolate ~~membrance~~ associated and secreted extracellular proteins from ''Streptomyces''. Developed by Dave Widdick<sup>[1]</sup>, the protocol uses Lithium Chloride to selectively solubilize cell surface proteins<sup>[REF]</sup		This protocol can be used to isolate membrane associated and secreted extracellular proteins from ''Streptomyces''. Developed by Dave Widdick<sup>[1]</sup>, the protocol uses Lithium Chloride to selectively solubilize cell surface proteins<sup>[REF]</sup
	>, and trichloroacetic acid to precipitate the proteins from solution for purification and concentration. Cells are grown on cellophane disks so that they can be easily scraped off of plates, and to reduce medium contamination of the samples.		>, and trichloroacetic acid to precipitate the proteins from solution for purification and concentration. Cells are grown on cellophane disks so that they can be easily scraped off of plates, and to reduce medium contamination of the samples.

Sam Prudence at 16:01, 12 September 2019

2019-09-12T16:01:58Z

← Older revision		Revision as of 16:01, 12 September 2019
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	# Pellet biomass via centrifugation at 1800g for 5 minutes, then pass the supernatant through a 0.45µm filter into a fresh tube. Filtering should minimise cellular contamination, low protein affinity cellulose acetate filters can be used here to minimise protein loss.		# Pellet biomass via centrifugation at 1800g for 5 minutes, then pass the supernatant through a 0.45µm filter into a fresh tube. Filtering should minimise cellular contamination, low protein affinity cellulose acetate filters can be used here to minimise protein loss.
	# Precipitate proteins from filtered supernatant by adding TCA to 20% and incubating samples on ice for 30 minutes or overnight.		# Precipitate proteins from filtered supernatant by adding TCA to 20% and incubating samples on ice for 30 minutes or overnight.
	# If you are also sampling the cellular fraction, at this stage the cell pellets can be suspended in your [[Cell lysis buffer]] of choice and lysed via your method of choice (e.g. sonication, boiling at 100°C, see [[Cell lysis buffer]] for advice on ''Streptomyces'' lysis.		# If you are also sampling the cellular fraction, at this stage the cell pellets can be suspended in your [[Cell lysis buffer]] of choice and lysed via your method of choice (e.g. sonication, boiling at 100°C, see [[Cell lysis buffer]] for advice on ''Streptomyces'' lysis buffers and methods).
	# After incubation on ice, pellet the precipitated protein by spinning at 1800g for 15 minutes (Note: it is recommended that a cooling centrifuge is used at 4°C).		# After incubation on ice, pellet the precipitated protein by spinning at 1800g for 15 minutes (Note: it is highly recommended that a cooling centrifuge is used at 4°C, keeping samples cool will minimise protein degredation).
	# If this generated two liquid phases, remove the upper phase and discard, then add dH<sub>2</sub>O to bring the lower phase to the original volume.		# If this generated two liquid phases, remove the upper phase and discard, then add dH<sub>2</sub>O to bring the lower phase to the original volume.
	# Pellet protein via centrifugation at full speed for 15 minutes		# Pellet protein via centrifugation at full speed for 15 minutes.
	# Wash the pellet three times with -20°C acetone and air dry (Note: you do not need to suspend your protein pellet in acetone to wash the pellet! Splashing the pellet with 0.5-1ml ice cold acetone, then re-pelleting, will be sufficient).		# Wash the pellet three times with -20°C acetone and air dry (Note: you do not need to suspend your protein pellet in acetone to wash the pellet! Splashing the pellet with 0.5-1ml ice cold acetone, then re-pelleting, will be sufficient).
	# For long term storage place the pellet at -80°C.		# For long term storage place the pellet at -80°C.
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	==Notes==		==Notes==
	*Re-suspending TCA precipitated protein can be highly challenging! SDS and gentle heating is recommended.		*Re-suspending TCA precipitated protein can be highly challenging! SDS and gentle heating is recommended.
	*If you wish to perform a Bradford Assay, urea is recomended to aid re-suspension.		*If you wish to perform a Bradford Assay, urea is recomended instead of SDS to aid re-suspension.

Sam Prudence: /* Isolation of extracellular protein from Streptomyces */

2019-09-12T15:58:38Z

Isolation of extracellular protein from Streptomyces

← Older revision		Revision as of 15:58, 12 September 2019
Line 1:		Line 1:

	=Isolation of extracellular protein from ''Streptomyces''=		=Isolation of extracellular protein from ''Streptomyces''=
	This protocol can be used to isolate membrance associated and secreted extracellular proteins from ''Streptomyces''. ~~Based on work~~ by Dave Widdick<sup>[1]</sup>, the protocol uses Lithium Chloride to selectively solubilize cell surface proteins<sup>[REF]</sup		This protocol can be used to isolate membrance associated and secreted extracellular proteins from ''Streptomyces''. Developed by Dave Widdick<sup>[1]</sup>, the protocol uses Lithium Chloride to selectively solubilize cell surface proteins<sup>[REF]</sup
	>, and trichloroacetic acid to precipitate the proteins from solution for purification and concentration. Cells are grown on cellophane disks so that they can be easily scraped off of plates, and to reduce medium contamination of the samples.		>, and trichloroacetic acid to precipitate the proteins from solution for purification and concentration. Cells are grown on cellophane disks so that they can be easily scraped off of plates, and to reduce medium contamination of the samples.

	==Buffers & Materials==		==Buffers & Materials==
	*Solid medium of choice (Note: for LC MS based proteomics, media with peptone, tryptone, or raw extracts such as yeast extract should be avoided, in these cases [[R2]] or [[MM]] are recommended)		*Solid medium of choice (Note: for LC MS based proteomics media with peptone, tryptone, or raw extracts such as yeast extract should be avoided, in these cases [[R2]] or [[MM]] are recommended)
	*Sterile cellophane disks		*Sterile cellophane disks
	*5M Lithium Chloride (LiCl) solution		*5M Lithium Chloride (LiCl) solution

Sam Prudence: Created page with " =Isolation of extracellular protein from ''Streptomyces''= This protocol can be used to isolate membrance associated and secreted extracellular proteins from ''Streptomyces''..."

2019-09-12T15:57:16Z

Created page with " =Isolation of extracellular protein from ''Streptomyces''= This protocol can be used to isolate membrance associated and secreted extracellular proteins from ''Streptomyces''..."

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=Isolation of extracellular protein from ''Streptomyces''=
This protocol can be used to isolate membrance associated and secreted extracellular proteins from ''Streptomyces''. Based on work by Dave Widdick[1], the protocol uses Lithium Chloride to selectively solubilize cell surface proteins[REF], and trichloroacetic acid to precipitate the proteins from solution for purification and concentration. Cells are grown on cellophane disks so that they can be easily scraped off of plates, and to reduce medium contamination of the samples.

==Buffers & Materials==
*Solid medium of choice (Note: for LC MS based proteomics, media with peptone, tryptone, or raw extracts such as yeast extract should be avoided, in these cases [[R2]] or [[MM]] are recommended)
*Sterile cellophane disks
*5M Lithium Chloride (LiCl) solution
*100% Trichloroacetic Acid (TCA) solution
*[[Cell lysis buffer]] of choice
*Acetone
*0.45µm filters

==Protocol==

# Grow desired strains on sterile [[cellophane disks]] on medium of choice.
# Scrape the biomass off of the cellophane disk using a sterile spatula and suspend in ice cold 5M LiCl solution, then vortex for 2-3 minutes.
# Pellet biomass via centrifugation at 1800g for 5 minutes, then pass the supernatant through a 0.45µm filter into a fresh tube. Filtering should minimise cellular contamination, low protein affinity cellulose acetate filters can be used here to minimise protein loss.
# Precipitate proteins from filtered supernatant by adding TCA to 20% and incubating samples on ice for 30 minutes or overnight.
# If you are also sampling the cellular fraction, at this stage the cell pellets can be suspended in your [[Cell lysis buffer]] of choice and lysed via your method of choice (e.g. sonication, boiling at 100°C, see [[Cell lysis buffer]] for advice on ''Streptomyces'' lysis.
# After incubation on ice, pellet the precipitated protein by spinning at 1800g for 15 minutes (Note: it is recommended that a cooling centrifuge is used at 4°C).
# If this generated two liquid phases, remove the upper phase and discard, then add dH2O to bring the lower phase to the original volume.
# Pellet protein via centrifugation at full speed for 15 minutes
# Wash the pellet three times with -20°C acetone and air dry (Note: you do not need to suspend your protein pellet in acetone to wash the pellet! Splashing the pellet with 0.5-1ml ice cold acetone, then re-pelleting, will be sufficient).
# For long term storage place the pellet at -80°C.

==Notes==
*Re-suspending TCA precipitated protein can be highly challenging! SDS and gentle heating is recommended.
*If you wish to perform a Bradford Assay, urea is recomended to aid re-suspension.

Protocol adapted from Widdick ''et al.''[1] by Sam Prudence, University of East Anglia.

==References==
# Widdick, D.A., Dilks, K., Chandra, G., Bottrill, A., Naldrett, M., Pohlschroder, M., Palmer, T. (2006). The twin-arginine translocation pathway is a major route of protein export in ''Streptomyces coelicolor''. ''Proceedings of the National Academy of Science'', 103(47), pp. 17927–17932 DOI: 10.1073/pnas.0607025103