Protein expression and purification - Revision history

ESO wikiadmin at 13:43, 5 June 2025

2025-06-05T13:43:44Z

← Older revision		Revision as of 15:43, 5 June 2025
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			''This is an original excerpt from SBAs digital labbook from May 2025.''

	== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==		== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

ESO wikiadmin: /* Concentration Determination of purified recombinant hPOPX2 (PPM1F) */

2025-06-05T13:42:42Z

Concentration Determination of purified recombinant hPOPX2 (PPM1F)

← Older revision		Revision as of 15:42, 5 June 2025
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	== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==		== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
	~~- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)~~

	~~o Pipette~~ 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate		* Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)
			** Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate
	~~o Add~~ 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)		** Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)
			** Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.
	~~o Cover~~ plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.		** Measure absorbance at 660 nm
			** Perform standard curve and calculate the concentration of the protein sample
	~~o Measure~~ absorbance at 660 nm

	~~o Perform~~ standard curve and calculate the concentration of the protein sample


	'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer		'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer
			[[File:SBA curve.png\|center\|frameless\|604x604px]]

ESO wikiadmin: /* Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26 */

2025-06-05T13:41:04Z

Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26

← Older revision		Revision as of 15:41, 5 June 2025
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	== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==		== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
			[[File:SBA 25.png\|right\|frameless\|243x243px]]
	* Mix samples with 2x SDS loading buffer		* Mix samples with 2x SDS loading buffer
	* Boil samples 95°C for 5 min		* Boil samples 95°C for 5 min

ESO wikiadmin: /* Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2 */

2025-06-05T13:39:36Z

Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2

← Older revision		Revision as of 15:39, 5 June 2025
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	== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==		== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
			[[File:SBA 24.png\|right\|frameless\|389x389px]]
	* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)		* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
	* The column is hung upside down		* The column is hung upside down
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	* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here		* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
	* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1		* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
	* ~~[[File:SBA 24.png\|right\|frameless\|389x389px]]~~Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2		* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
	* Wash column with elution buffer 2 for 15 min at 0.5 mL/min		* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
	* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page		* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

ESO wikiadmin: /* Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26 */

2025-06-05T13:39:22Z

Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26

← Older revision		Revision as of 15:39, 5 June 2025
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	* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here		* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
	* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1		* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
	* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2		* [[File:SBA 24.png\|right\|frameless\|389x389px]]Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
	* Wash column with elution buffer 2 for 15 min at 0.5 mL/min		* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
	* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page		* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
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	== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==		== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
	~~- Mix samples with 2x SDS loading buffer~~

	~~- Boil~~ samples 95°C for 5 min		* Mix samples with 2x SDS loading buffer
			* Boil samples 95°C for 5 min
	~~- Load~~ onto a 12.5% acrylamide gel		* Load onto a 12.5% acrylamide gel
			* Run at 80 V/120 V
	~~- Run~~ at 80 V/120 V		* Coomassie stain after short heating in microwave for 40 min
			* Destain until bands become visible
	~~- Coomassie~~ stain after short heating in microwave for 40 min

	~~- Destain~~ until bands become visible

	== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==		== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==

ESO wikiadmin at 13:37, 5 June 2025

2025-06-05T13:37:29Z

← Older revision		Revision as of 15:37, 5 June 2025
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	* Destain until bands are visible		* Destain until bands are visible

	''Cleaning of Column after use:''		''Cleaning of Column after use:''

	~~- wash~~ 10 min 1 mL/min with Elutionbuffer 2 (10 mL)		* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
			* wash 20 min 1 mL/min with Wash buffer (20 mL)
	~~- wash~~ 20 min 1 mL/min with Wash buffer (20 mL)		* wash 20 mL 1 mL/min with H2O (degassed)
			* wash 20 mL 20% EtOH
	~~- wash~~ 20 mL 1 mL/min with H2O (degassed)

	~~- Wash~~ 20 mL 20% EtOH

	== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==		== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==

ESO wikiadmin at 13:37, 5 June 2025

2025-06-05T13:37:00Z

← Older revision		Revision as of 15:37, 5 June 2025
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	== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==		== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
	~~- Incubate~~ 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample		Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

	== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==		== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
	~~- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)~~

	~~- The~~ column is hung upside down		* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
			* The column is hung upside down
	~~- Pump~~ water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)		* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
			* Remove the tube from the end of the column and insert column the right way round
	~~- Remove~~ the tube from the end of the column and insert column the right way round		* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
			* Prepare 6 Schnappdeckelröhrchen
	- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL		* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
			* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
	~~- Prepare~~ 6 Schnappdeckelröhrchen		* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
			* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
	~~- Prepare~~ 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)		* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
			* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
	~~- Pump~~ sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here		* Run SDS-Page at 80V /120 V
			* Stain gel with coomassie for 45 min at rt after short warming in microwave
	~~- Start~~ with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1		* Destain until bands are visible

	~~- Switch~~ to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

	~~- Wash~~ column with elution buffer 2 for 15 min at 0.5 mL/min

	~~- Mix~~ 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

	~~- Run~~ SDS-Page at 80V /120 V

	~~- Stain~~ gel with coomassie for 45 min at rt after short warming in microwave

	~~- Destain~~ until bands are visible

	''Cleaning of Column after use:''		''Cleaning of Column after use:''

ESO wikiadmin at 13:35, 5 June 2025

2025-06-05T13:35:33Z

← Older revision		Revision as of 15:35, 5 June 2025
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	== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==		== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==
	~~- Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub> (no glycerin!)~~

	- Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub> (no glycerin!)		* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub> (no glycerin!)
			* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub> (no glycerin!)
			* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub> (no glycerin!)
			* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

	~~- Dialysis in 1.5 L~~ for ~~4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl<sub>2</sub>~~ (~~no glycerin!~~)		== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
			[[File:SBA 21.png\|right\|frameless\|

	~~- Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C~~		*

	~~== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==~~
	~~- Dialysis performed by SF~~

	~~- Mix samples with 2x SDS loading buffer~~		]]

	~~- Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel~~		* Dialysis performed by SF
			* Mix samples with 2x SDS loading buffer

	~~- Perform SDS-Page at 80V/120V~~		* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

	- ~~Coomassie stain gel after short heating in the microwave for 30 min~~ at rt		* Perform SDS-Page at 80V/120V

	~~- Destain~~ until bands are visible		* Coomassie stain gel after short heating in the microwave for 30 min at rt
			* Destain until bands are visible

	'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2		'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

ESO wikiadmin at 13:32, 5 June 2025

2025-06-05T13:32:54Z

← Older revision		Revision as of 15:32, 5 June 2025
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	== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==		== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
	~~[[File:SB20.png\|right\|frameless]]~~
	Lysis of bacteria:		Lysis of bacteria:

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	== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==		== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
			[[File:SB20.png\|right\|frameless\|328x328px]]
	* Purification of recombinant PPM1F via HisSUMO tag conducted by SF		* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
	* Mix samples with 2x SDS loading buffer		* Mix samples with 2x SDS loading buffer

ESO wikiadmin at 13:31, 5 June 2025

2025-06-05T13:31:57Z

← Older revision		Revision as of 15:31, 5 June 2025
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	== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==		== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
			[[File:SB20.png\|right\|frameless]]
	Lysis of bacteria:		Lysis of bacteria: