Difference between revisions of "Part:BBa K4439007"

Revision as of 22:16, 11 October 2022 (view source) Charlottedml (Talk | contribs) ← Older edit		Revision as of 22:22, 11 October 2022 (view source) Charlottedml (Talk | contribs) Newer edit →
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	==Experiments==		==Experiments==
−	===Adapted Protocols===	+	===Protein Purification Protocol using Ni-NTA Beads===
−	====Protein Purification using Ni-NTA Beads====	+	====Materials====
−		+
−	=====Materials=====	+
	*2.5M imidazole stock solution		*2.5M imidazole stock solution
Line 49:		Line 47:
	*Tube of bacterial culture induced with IPTG		*Tube of bacterial culture induced with IPTG
−	=====Procedure=====	+	====Procedure====
−	======Step 1: Prepare Buffers======	+	=====Step 1: Prepare Buffers=====
	*Wash Buffer A (1L)		*Wash Buffer A (1L)
Line 60:		Line 58:
	. 20 mM imidazole (for non specific binding)		. 20 mM imidazole (for non specific binding)
		+
		+	*Elution Buffer B (250 mL)
		+
		+	. 300 mM NaCl
		+
		+	. 20 mM HEPES pH 7.5
		+
		+	. 500 mM imidazole pH 7.5
		+
		+	* We use stocks of 5M NaCl, 1M HEPES pH 7.5 and 2.5M imidazole pH 7.5.
		+
		+	=====Step 2: Preparation of our sample=====
		+
		+	*Defrost your tube of pellet in water, or obtain a cell pellet by centrifugation at 5000xg for 10min at 4°C.
		+
		+	*Dilute the pellet with the wash buffer (ideally we need big volumes). We resuspent our cell pellet into 20mL. Bigger the volume is, the better. However, be careful to not completely drown the pellet with the wash buffer.
		+
		+	*Add some bonus stuff which are useful :
		+
		+	. Add 1 protease inhibitor tablet. As previously, just approximate and don’t pipette it.
		+	Protease inhibitor protects the proteins sensitive to degradation and inhibit their destruction. Quite expensive.
		+
		+	. Add 5 ul of turbonuclease. Pipette the precise volume.
		+	Turbonuclease cleaves DNA to avoid the formation of jelly that is due to the presence of undestroyed DNA or RNA. Therefore, it ensures a better fluidity for our sample.
		+
		+	*Mix well and vortex quickly, it makes the sample more homogenous. We can be harsh to cells without fragilising them.
		+	WARNING : After this step do not vigorously shake your sample !
		+	After cell lysis, proteins are not within the protective environment of the cell.
		+
		+	*Put the cultures on a shaker at 4°c for 20min.
		+
		+	*Lyse cells with a sonicator at 70% power, 10 secs on and 10 secs off pulse cycle over 2.5 minutes (in total, set the machine for 5 minutes). The samples will get hot. Then, put the sample on ice.
		+	Place the sample on ice in a beaker. Wash the tip with alcohol and water before and after.
		+	Sonication uses sound waves to explode the cells.
		+
		+	*Centrifuge the lysed cells (lysate) at 20 000 x g for 30 minutes (1 hour or 2 hours could still be fine).
		+	For the balances for the centrifuge, take a flask and fill it approximately with the same volume of the sample with water.
		+	To open the machine, push the cover and pull it out. Stay next to the machine till it shows that it has reached the wanted speed. The sample should be less viscous after the centrifugation.

---

Revision as of 22:22, 11 October 2022

mSA-N[AS]4C-CBD-10xHis

Abstract

To complete

Sequence and Features

Protein's Characterization

Description

To complete

Usage and Biology

To complete

Modeling

To complete

Experiments

Protein Purification Protocol using Ni-NTA Beads

Materials

• 2.5M imidazole stock solution

• 5M NaCl solution

• Protease inhibitor tablet

• Turbonuclease

• Sonicator

• 1M HEPES

• HisPur Ni-NTA beads in 20% ethanol

• Disposable plastic column

• Centrifuge going to 20 000 xg

• 0.30 µm filters

• Tube of bacterial culture induced with IPTG

Procedure

Step 1: Prepare Buffers

• Wash Buffer A (1L)

. 300 mM NaCl

. 20 mM HEPES pH 7.5

. 20 mM imidazole (for non specific binding)

• Elution Buffer B (250 mL)

. 300 mM NaCl

. 20 mM HEPES pH 7.5

. 500 mM imidazole pH 7.5

• We use stocks of 5M NaCl, 1M HEPES pH 7.5 and 2.5M imidazole pH 7.5.

Step 2: Preparation of our sample

• Defrost your tube of pellet in water, or obtain a cell pellet by centrifugation at 5000xg for 10min at 4°C.

• Dilute the pellet with the wash buffer (ideally we need big volumes). We resuspent our cell pellet into 20mL. Bigger the volume is, the better. However, be careful to not completely drown the pellet with the wash buffer.

• Add some bonus stuff which are useful :

. Add 1 protease inhibitor tablet. As previously, just approximate and don’t pipette it. Protease inhibitor protects the proteins sensitive to degradation and inhibit their destruction. Quite expensive.

. Add 5 ul of turbonuclease. Pipette the precise volume. Turbonuclease cleaves DNA to avoid the formation of jelly that is due to the presence of undestroyed DNA or RNA. Therefore, it ensures a better fluidity for our sample.

• Mix well and vortex quickly, it makes the sample more homogenous. We can be harsh to cells without fragilising them.

WARNING : After this step do not vigorously shake your sample ! After cell lysis, proteins are not within the protective environment of the cell.

• Put the cultures on a shaker at 4°c for 20min.

• Lyse cells with a sonicator at 70% power, 10 secs on and 10 secs off pulse cycle over 2.5 minutes (in total, set the machine for 5 minutes). The samples will get hot. Then, put the sample on ice.

Place the sample on ice in a beaker. Wash the tip with alcohol and water before and after. Sonication uses sound waves to explode the cells.

• Centrifuge the lysed cells (lysate) at 20 000 x g for 30 minutes (1 hour or 2 hours could still be fine).

For the balances for the centrifuge, take a flask and fill it approximately with the same volume of the sample with water. To open the machine, push the cover and pull it out. Stay next to the machine till it shows that it has reached the wanted speed. The sample should be less viscous after the centrifugation.

Lab's Tips and Tricks

To complete

Results

Expression

To complete

Purification

To complete

Biofilm Fabrication

To complete

Hydrophobicity tests

To complete

References