Mar 03, 2020

Public workspaceProtein extraction form Aurantiochytrium limacinum (ATCC MYA-1381)

DOI
dx.doi.org/10.17504/protocols.io.bc7gizjw
  • 1University of Illinois at Urbana-Champaign
  • Collier Lab
Anbarasu Karthikaichamy
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DOI: dx.doi.org/10.17504/protocols.io.bc7gizjw
Protocol Citation: Anbarasu Karthikaichamy 2020. Protein extraction form Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.bc7gizjw
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: March 03, 2020
Last Modified: March 03, 2020
Protocol Integer ID: 33736
Keywords: Stramenopile, Thraustochytrid, Aurantiochytrium, Protists, protein extraction,
Abstract
Protein extraction protocol for Aurantiochytrium limacinum (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid).


Safety warnings
The lysis buffer contains detergents, and therefore it should be handled with care. Always wear gloves to minimize keratin contamination.
Before start
Get a bucket full of ice, and always keep the lysed culture or protein suspension on ice.
Lysis buffer preparation
Lysis buffer preparation
10m
10m
Start by making stocks of the following,

  • Concentration1 Molarity (M) KCl
  • Concentration25 millimolar (mM) MgCl2
  • Concentration1 Molarity (M) Tris

Prepare lysis buffer (LyB) by adding the following chemicals (for 10ml),

ChemicalVolume (uL)
KCl (1M)500
MgCl2 (25mM)1000
Tris (1M)500
NP40 (100%)45
Tween (100%)45
dH2O8310






10m
Cell harvesting
Cell harvesting
10m
10m
  • Pipette out ~3 ml of cell suspension from the test tube or conica flask.
  • Centrifuge the cells at Centrifigation3000 rpm, 4°C, 00:10:00
  • Discard the supernatant and keep the cell pellet on ice.

10m
Lysis and soluble fraction extraction
Lysis and soluble fraction extraction
20m
20m
  • Add 1 ml of lysis buffe (LyB) to the pellet.
  • Vortex the mixture for 20 mins on high spped.
  • Centrifuge the suspension at Centrifigation13500 rpm, 4°C, 00:15:00
  • Collect the supernatant in a separate tube and place it on ice. This is the soluble fraction.
20m
Insoluble fraction extraction
Insoluble fraction extraction
40m
40m
  • Resuspend the pellet from the above step in 4x LDS sample buffer
  • Add required amount of dH2O
  • Add 1:10(v:v) 25% β-Mercaptoethanol
Safety information
Always handle β-Mercaptoethanol in fume hood!
  • Heat the mixture at Temperature70 °C for Duration00:10:00
  • Centrifuge the mixture at Centrifigation13500 rpm, 4°C, 00:20:00
  • Carefully aspirate the supernatant (insoluble fraction) into a fresh tube and keep it on ice.

Note
Alternatively, one could also use custom buffers (with high concentration of detergent) for insoluble fraction extraction.


40m
Quantification and visualization
Quantification and visualization
Quantify the protein extract using an appropriate mehtod and the proteins can be visualized on a SDS-PAGE gel stained with coomassie blue.