Difference between revisions of "Part:BBa K4439007"
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===Protein Purification Protocol using Ni-NTA Beads===
 
===Protein Purification Protocol using Ni-NTA Beads===
  
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[[His-tagged Protein Purification:His-tagged Protein Purification.png]]
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[[His-tagged Protein Purification:His-tagged_Protein_Purification.png]]
  
 
====Materials====
 
====Materials====

Revision as of 22:39, 11 October 2022

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


Abstract

To complete

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Protein's Characterization

Description

To complete

Usage and Biology

To complete

Modeling

To complete

Experiments

Protein Purification Protocol using Ni-NTA Beads

His-tagged Protein Purification:His-tagged_Protein_Purification.png

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.

Step 3: Resin preparation
  • If needed, pour out a bit of ethanol from the Ni-NTA beads in solution.
  • Equilibrate 2-5 mL Ni-NTA beads into the wash buffer (this removes the 20% ethanol in which the beads are stored), 2 mL for low expressing protein constructs, 5 mL for high expressors.
  • Measure out 2-5 mL of settled beads (we measured 25 mL of the beads in ethanol which corresponded to 5 mL of settled beads)
  • Empty (as much as possible) the ethanol without letting go of beads.
  • Repeat 3 times :

.Add the wash buffer to around 40-50 mL, mix a bit.

.Centrifuge the tube at 500g for 5 minutes (do not forget to balance the centrifuge with a tube of water).

.Empty (as much as possible) the supernatant.

Step 4: Incubation of lysate with Ni-NTA resin
  • When the centrifuge is finished, carefully transfer the supernatant into a beaker on ice. Keep some in the tube for the SDS-PAGE.
  • With a syringe, aspirate the supernatant CAREFUL NOT TO MAKE BUBBLES.
  • If you are using a machine : filter the buffers using a 0.45 µm filter.
  • Add imidazole to the filtered supernatant to 25 mM. This reduces non-specific binding of endogenous His-rich E.coli proteins.
  • Decant the excess liquid from the washed beads.
  • Add the supernatant to the beads.
  • Put the beads with the supernatant rotating overnight at 4°C.
Step 5: Purification
  • Setup a stand with a plastic disposable column.
  • Transfer the beads to a disposable plastic column.
  • Wash the beads in sequential order to remove loosely bound proteins before elution. The washes contain increasing amounts of imidazole to remove unbound, unspecific proteins from the resin. Do not forget to label everything and to keep everything to know where the proteins are. Keep all your fractions on ice.

. Wash 1 - 20 mM imidazole - 30 mL

. Wash 2 - 50 mM imidazole - 30 mL

. Elute 1 - 250 mM imidazole - 30 mL

. Elute 2 - 500 mM imidazole - 30 mL

.If needed:

.Elute 3 - 1 M imidazole - 20 mL

.Elute 4 - 2.5 M imidazole - 10 mL

.Elute 5 - 5 M imidazole - 10 mL

  • We had to do more elution steps than planned because the control (GFP protein) which contains both the CBD domains and the mSA didn’t eluted at the normal elution concentration.

In what fraction the protein elutes depends on the size of the His-tag, where the tag is (N or C) and the 3D context of the target (is the tag accessible to the resin).

Step 6: Concentration
  • Concentrate the protein using a filter of 30kDa.
  • Try to remove the imidazole excess by puting the concentrated proteins under dialysis. The dialysis buffer was composed of 300mM NaCl and 20 mM HEPES.
  • To see if there are protein aggregations, we centrifuged the result after dialysis. If a pellet appears, it means that there is an aggregation of protein which may mean that the proteins are not functional anymore.
  • Finally we measured the concentration of the protein after dialysis.


Lab's Tips and Tricks

To complete

Results

Expression

To complete

Purification

To complete

Biofilm Fabrication

To complete

Hydrophobicity tests

To complete

References