Dec 12, 2022

Chloroform-methanol protein precipitation from microalgae and Pierce BCA assay

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm2e25g3p/v1
Chloroform-methanol protein precipitation from microalgae and Pierce BCA assay
  • 1Dalhousie University
Yingyu Hu
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm2e25g3p/v1
Protocol Citation: Ying-Yu Hu, Christopher Lord, Zoe V Finkel 2022. Chloroform-methanol protein precipitation from microalgae and Pierce BCA assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2e25g3p/v1
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: November 28, 2022
Last Modified: December 12, 2022
Protocol  Integer ID: 73284
Keywords: Protein , Microalgae, Chloroform-methanol precipitation, Pierce BCA assay, methanol protein precipitation from microalgae, methanol protein precipitation, bca assay, pierce bca protein, extracted protein, methanol, chloroform, detergent in crude protein, crude protein, protein, chlorophyll, microalgae, phospholipid
Funders Acknowledgements:
Simons Collaboration on Computational Biogeochemical Modeling of Marine Ecosystems
Grant ID: 549937
Ocean Processes and Ecology
Grant ID: 723789
Abstract
Chlorophyll, phospholipids, sucrose, glycerol and some detergent in crude protein extracted from microalgae can interfere the Pierce BCA protein assay. In order to remove these interference, bead miller extracted protein is precipitated by chloroform-methanol prior to BCA assay. The resulting precipitation is dissolved into Sarcosine-Tris solution. Low limit of detection is about 5 ug/mL.
Protocol materials
Tris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
N-lauroylsacosine sodium salt solution (30%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #61747
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860
Chloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)Thermo ScientificCatalog #Thermo Scientific™ 0023210
Pierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
Safety warnings
Use fume-hood when handling methanol and chloroform.

All waste containing methanol and chloroform shall be collected in waste container for halogenated organic solvents.
Reagent preparation
Tris buffer 5 Mass Percent (pH 8.0)
Add 500 µL 1 Mass Percent 8.0 Tris into 100 mL MilliQ waterTris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
20% Sarcosine
Dilute 2 part 30% N-lauroylsarcosine sodium salt with 1 part 5 Mass Percent (pH 8.0) Tris buffer
N-lauroylsacosine sodium salt solution (30%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #61747
Protein precipitation
1h 12m
Thaw protein extract
Turn on refrigerate centrifuge
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU

Turn on incubator/shaker, preheat to 37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

Prepare ice-bath
Well mix the extract and then transfer 100 µL of extract to 2 mL microtube (Abdos tubes give better precipitation results), in replicate.

Equipment
Micro Centrifuge Tubes
NAME
Abdos
BRAND
P10203
SKU

Note
If extract has debris, spin down debris by 13300 rpm, Room temperature, 00:05:00 and transfer only clear supernatant.
Debris can cause overestimation of protein content.


In the fume hood, add 400 µL methanol
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860
Gently vortex for 00:00:30 by using a tube insert
Equipment
VWR ANALOG VORTEX MIXER
NAME
VWR
BRAND
10153-838
SKU
With tube insert
SPECIFICATIONS

30s
In the fume hood, add 100 µL chloroformChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
Gently vortex for 00:00:30 by using a tube insert
30s
In the fume hood, add 300 µL MilliQ
Gently vortex for 00:00:30 by using a tube insert
30s
Incubate On ice for 00:30:00

30m
20000 rcf, 4°C, 00:10:00
10m
In the fume hood, remove upper phase by leaving about 250 µL liquid
Note
Do not disturb the interphase

In the fume hood, add 300 µL methanol
Gently mix the liquid until bottom layer disappear and the solution is homogenous.
Note
The formation of small pellet might be observed, but might be invisible due to low protein mass.

20000 rcf, 4°C, 00:10:00
10m
In the fume hood, remove all solvent.
Note
Watch the pipet closely. Do not remove pellets with the solvent.

If pellet tends to be aspired with solvent, add another 300 µL methanol, gently vortex, and 20000 rcf, 4°C, 00:10:00

10m
In the fume hood, remove most solvent by using 1000 uL pipet tip, and then remove the rest by using 100 uL pipet tip. Do not remove pellet with solvent.
Dry pellet in vacuum desiccator for at least 00:30:00 at Room temperature
Note
Any methanol and chloroform residue can affect the re-dissolving of pellet in BCA assay.
However, do not dry protein pellet for too long, otherwise it might be difficult to re-dissolve.

30m
BCA assay
30m
Add 5 µL 20% sarcosine and 95 µL 5 Mass Percent (pH 8.0) Tris buffer to dry protein pellet, incubate at 37 °C for 15 to 30 min.

Use tube insert, vortex all tubes for 15 to 30 min until pellet is completely re-dissolved.
BSA standard solutions
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)Thermo ScientificCatalog #Thermo Scientific™ 0023210
Standard20% sarcosine (uL)5 mM Tris (uL)2 mg/mL BSA (uL)Final Conc. (mg/mL)
SD159500
SD22547050.02
SD325463120.048
SD425450250.1
SD525425500.2
SD6253751000.4
SD7252752000.8
SD8252252501

Vortex and then use reverse pipetting: transfer 100 µL standard solutions into the corresponding tubes, except for SD1 (it has already been 100 uL).
Use the following formula to determine the total volume of working reagent (WR) required. Consider leaving several mL of extra volume:

(# standards + # samples) X (800 µL ) = total volume WR required
Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube

Pierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
Use one tip and reverse pipetting: Add 800 µL WR into each tube, make sure that the tip doesn't have contact with the solution, so that samples are not cross-contaminated.
Note
Since BCA assay is sensitive to duration, although reagent is aqueous, it is more efficient to use reverse pipetting and quickly dispense reagent into all tubes, therefore the duration difference amongst standards and samples can be minimized.

Vortex each tube, shake and incubate at 37 °C for 00:30:00
30m
Remove samples from the incubator.
Load samples into microplate in duplicate:
Note
  1. Reverse pipetting: aspire 200 µL sample from the middle of solution
  2. Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
  3. Dispose the tip
  4. Use a new tip, reverse pipet another 200 µL as replicate
  5. Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate

Equipment
96-Well Microplates
NAME
Polystyrene, Clear,
TYPE
Greiner Bio-One
BRAND
82050-760
SKU

Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 562 nm with a measurement time 100 ms
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Calculation
Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard.
Subtract the average 562 nm absorbance measurement of the blank sample (filter) replicates from the 562 nm measurements of all other individual sample.
Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard versus its concentration in mg/ml. The standard curve is quadratic.

For the calculation convenience, plot BSA concentration (Conc) versus Corrected absorbance (Abs) to obtain a standard curve as following:
Conc_mg/mL = a X Abs^2 + b X Abs + c

Use the corrected measured absorbance of samples (Abs) to calculate the total protein concentration (Conc_mg/mL) from each sample.
Protein_mg/filter = Conc_mg/mL X PEB_mL
Where PEB is the volume of protein extraction buffer used to extract protein from microalgae sample.