Sep 08, 2018

Public workspaceExtraction and Lowry-Assay for determination of Synechocystis total protein

DOI
dx.doi.org/10.17504/protocols.io.taseiee
  • 1Heinrich-Heine Universität Düsseldorf;
  • 2Institute for Synthetic Microbiology, HHU Düsseldorf
  • Axmann Lab
  • CyanoWorld
Sebastian ST Triesch
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DOI: dx.doi.org/10.17504/protocols.io.taseiee
Protocol Citation: Anika Wiegard, Sebastian ST Triesch 2018. Extraction and Lowry-Assay for determination of Synechocystis total protein. protocols.io https://dx.doi.org/10.17504/protocols.io.taseiee
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 06, 2018
Last Modified: September 08, 2018
Protocol Integer ID: 15410
Keywords: Synechocystis, Protein, Lowry
Abstract
A quick Lowry.Assay for the extraction and determination of total protein from Synechocystis.
Photo credit: Miriam Dreesbach, Institute for Synthetic Microbiology, HHU Düsseldorf
Materials
MATERIALS
ReagentTrichloroacetic acidP212121
ReagentSodium Hydroxide Thermo Fisher ScientificCatalog #S320
Reagent1kg Sodium Carbonate; Na2CO3 (anhydrous)G-BiosciencesCatalog #RC-126
ReagentCopper (II) sulfate pentahydrateBio Basic Inc.Catalog #CDB0063.SIZE.2.5Kg
ReagentFolin & Ciocalteu’s phenol reagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #F9252
ReagentPotassium sodium tartrate tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2377
Before start
Remeber to prepare BSA standards to absolutely quantify your extracted total protein. A BSA range of 5 µg/ml to 100 µg/ml BSA in water or appropiate media showed good results. 
Sample 1 ml of your Synechocystis culture in 2 ml Eppendorf tubes.
Add 110 µl of 100% trichloracetic acid and incubate the mixture on ice for 20 min. 
Duration00:20:00 Incubation on ice
Centrifuge your mixture for 10 min at 15,000 g at 4 °C.
Duration00:10:00 Centrifugation
Discard the supernatant thoroughly and place the tubes upright for 10 min. Tap the upright tubes carefully until all liquid is removed. 
Resuspend the pellet carefully in 500 µl of a 1 M NaOH solution. Vortex and incubate the samples over night (approx. 16 hours) at room temperature. 
Prepare a Lowry solution by mixing the following reagents in the given order:
  • 500 µl K-Na-tartrate (2%)
  • 500 µl Cu2SO4*5 H2O (1%)
  • 100 ml Na2CO3 (2%)
Scale the total volumes down to an appropriate amount. You will need 900 µl Lowry-Mix for each sample. Mix the Lowry-solution on the same day you use it and store it in the fridge in the meantime. 
In a new 1.5 ml Eppendorf tube, mix 100 µl sample (in NaOH) and 900 µl Lowry-Mix.
Add 100 µl 50% Folin & Ciocalteu's phenol reagent (diluted in water). Immediately incubate in the dark for 45 min. 
Duration00:45:00 Incubation in the dark
Spin down your incubated solution at 14,000 g for 5 min to remove lipids and cell debris. 
Duration00:05:00 Centrifugation
Carefully transfer 1 ml of your Sample-Lowry-Folin-Mix into a plastic cuvette and measure extinction at 750 nm. 
For absolute quantification, prepare a standard BSA concentration range from 5 to 100 µg/ml, and follow all steps above with your standard sample. 
Go to BSA standard set