Mar 04, 2020

Public workspaceTCA protein extraction from diatoms V.2

DOI
dx.doi.org/10.17504/protocols.io.bc7rizm6
  • 1Stazione Zoologica Anton Dohrn Napoli - Italy;
  • 2Stazione Zoologica Anton Dohrn
  • Metabolomics Protocols & Workflows
    Tech. support email: bbmisraccb@gmail.com
Anna Santin
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DOI: dx.doi.org/10.17504/protocols.io.bc7rizm6
Protocol Citation: Anna Santin, Antonella Ruggiero, Francesco Manfellotto, Mariella Ferrante 2020. TCA protein extraction from diatoms. protocols.io https://dx.doi.org/10.17504/protocols.io.bc7rizm6
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 04, 2020
Last Modified: March 04, 2020
Protocol Integer ID: 33745
Abstract
Protein extraction from diatoms using TCA
Materials
3X SDS Laemmli Buffer:

  • 240 mM Tris HCl (pH 6.8)
  • 6% SDS
  • 30% Glycerol
  • 2.28 M β-mercaptoethanol
  • 0.06% Bromophenol blue

Storerd: short term at 4°C
long term at -20°C
Pellet of around 50 ml of culture in exponential phase (2 million of cells)
Wash pellet with 1 ml of TCA 20%
Centrifuge at maximum speed for 1 minute
Pour off the supernatant and dilute in 100 µl of TCA 20%
Add Glass-Beads till meniscus and vortex for 7 minutes
Add 400 µl of TCA 5% and transfer the supernatant in new eppendorf (1.5 ml)
Centrifuge at 3000 rpm for 10 minutes (chiarification phase)
Pour off supernatant by aspiration
(now quickly!)
Add 100 µl of 3X SDS Laemmli Buffer (see Materials) and vortex very well to dissolve
Add 50 µl Tris Base and vortex very well to dissolve
Boil at 100°C for 3 minutes
Centrifuge at maximum speed for 5 minutes
Transfer supernatant containing proteins in a new eppendorf
Put tubes on ice
Stock at -20°C