Sep 03, 2020

Public workspaceChlamydomonas reinhardtii cell wall proteins recrystallization 

This protocol is a draft, published without a DOI.
  • 1Ronin Institute
Joao Vitor Molino
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Protocol Citation: Joao Vitor Molino 2020. Chlamydomonas reinhardtii cell wall proteins recrystallization . protocols.io https://protocols.io/view/chlamydomonas-reinhardtii-cell-wall-proteins-recry-bkrmkv46
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2020
Last Modified: September 03, 2020
Protocol Integer ID: 41485
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Abstract
This protocols describe the steps required for the recrystalization of cell wall proteins from Chlamydomonas reinhardtii from perchlorate extractions.


Protocol based on:

CITATION
Goodenough, U. W. Gebhart, B. Mecham, R. Heuser, J. E. (1986). Crystals of the Chlamydomonas reinhardtii cell wall: Polymerization, depolymerization, and purification of glycoprotein monomers. Journal of Cell Biology.
LINK
10.1083/jcb.103.2.405

Guidelines
All steps described in this protocol are intended to be conducted in a research laboratory. Follow aseptic procedures.
Materials
STEP MATERIALS
ReagentAmicon Ultra-0.5 Centrifugal Filter Unit 30 KDaSigma AldrichCatalog #UFC5030
Protocol materials
ReagentAmicon Ultra-0.5 Centrifugal Filter Unit 30 KDaMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC5030
ReagentAmicon Ultra-0.5 Centrifugal Filter Unit 30 KDaMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC5030
Safety warnings
Sodium perchlorate is a oxidizing agent, make sure to read the hazard information.
Before start
Before start
Check the protocol "Chlamydomonas reinhardtii cell wall extraction with perchlorate"
The amount of recrystalized material varies, but in some results I was able to obtain 1% of the inicial cell mass as cell wall material.

Recrystalization can be performed by diafiltration of dialysis.

By Diafiltration
By Diafiltration
1h 30m
1h 30m
  1. Prepare Amount1 mL of cell wall proteins extract by perchlorate treatment of Amount1 x 10^9 cells with Amount1 mL of Concentration2 Molarity (M) sodium perchlorate
  2. Add Amount500 µL of sample in centrifugal filters with a 30 KDa cutoff (e.g. Amicon Ultra 0.5 Centrifugal filters)
  3. Centrifuge at Centrifigation14000 rcf, 25°C, 00:20:00
  4. Remove the flow-through and add enought sample to the max capacity of the filter. Repeat until all sample processed
  5. Concentrate the sample until Amount100 µL
  6. Add 400 uL of ddH20
  7. Centrifuge at Centrifigation14000 rcf, 25°C, 00:20:00
  8. Repeat 4-7
  9. Collect the precipitated material by inverting the membrane in a new collecting tube and centrifuge at Centrifigation1000 rcf, 25°C, 00:01:00
  10. Proceed to planned experiments.
ReagentAmicon Ultra-0.5 Centrifugal Filter Unit 30 KDaMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC5030

1h 30m
By Dialsysis
By Dialsysis
2d
2d
  1. Prepare membranes with 30 Kda for dialysis, by cutting a segment larger enough to hold Amount1 mL of sample, and rehydrating it in ddH20 Duration00:30:00 prior to use
  2. Prepare Amount1 mL of cell wall proteins extract by perchlorate treatment of Amount1 x 10^9 cells with Amount1 mL of Concentration2 Molarity (M) sodium perchlorate,
  3. Add the extract to the membrane adn close it
  4. Place the membrane inside a vessel containig Amount1 L of ddH20. (A magnetic stirring can be used, making sure the membrane is not hit by the magnet bar)
  5. Incubate for Duration06:00:00
  6. Change the Amount1 L of ddH20 and incubate DurationOvernight
  7. Retrieve the liquid inside the tube, ressuspending any precipitate material by pipetting and transfering it to a clean microcentrifugal tube.
  8. Spin Centrifigation20000 rcf, 25°C, 00:01:00 , a precipitate should be visible
  9. Proceed to planned experiments.
2d
Citations
Goodenough, U. W. Gebhart, B. Mecham, R. Heuser, J. E.. Crystals of the Chlamydomonas reinhardtii cell wall: Polymerization, depolymerization, and purification of glycoprotein monomers
10.1083/jcb.103.2.405