Feb 28, 2024
Version 1
Comprehensive Protocol for Total Protein Extraction, Precipitation, and BCA Assay Measurement from Plankton Samples V.1
Forked from Total crude protein in plankton: Pierce BCA protein assay (including the enhanced assay for low biomass)
This protocol is a draft, published without a DOI.
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu 2024. Comprehensive Protocol for Total Protein Extraction, Precipitation, and BCA Assay Measurement from Plankton Samples. protocols.io https://protocols.io/view/comprehensive-protocol-for-total-protein-extractio-c85wzy7e
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: February 12, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95126
Keywords: microalgae, total protein, Pierce BCA, bead mill cell disruption, microplate, zooplankton, chloroform-methanol precipitation, protein extraction buffer
Funders Acknowledgement:
Simons Foundation
Grant ID: 723789
Simons Foundation
Grant ID: 549937
Abstract
This protocol outlines an optimized method for extracting total protein from plankton samples, encompassing both phytoplankton and zooplankton. The procedure involves the use of lysing matrix tubes with beads of varying sizes (0.1 mm, 1.4 mm, and 4 mm), eight homogenizing cycles, and a protein extraction buffer composed of 2% SDS, 10% glycerol, 5 mM EDTA in 100 mM Tris (pH 7). The extracted crude protein is quantified using the Pierce BCA assay, either incubation at 37°C for 20 to 2000 ug/mL or at 60°C for 5 to 200 ug/mL, using bovine serum albumin (BSA) as the standard.
To enhance accuracy, the extracted protein undergoes precipitation using a chloroform and methanol solvent mixture, followed by redissolution in re-solubilization buffer composed of 1% sarcosine in 50 mM Tris (pH 7). BCA assay is then used to quantify the precipitated protein, with BSA as the standard. The absence of glycerol and the use of sarcosine instead of SDS enable a linear detection range of 5 to 2000 µg/mL in this BCA assay at 37°C incubation.
Guidelines
1. Comparison of protein yield between the previous method and the improved method. The "Ratio" column represents the protein obtained from the improved method divided by that from the previous method.
| Species | Condition | Ratio | |
| Synecchococus (MITS1220) | Exponential | 3.29 (0.45) | |
| L. fissa (CCMP2935) | Stationary | 2.87 (0.33) | |
| L. fissa (CCMP2935) | Decline | 2.23 (0.18) | |
| Zooplankton (size: 500 - 2k um) | field sample | 1.95 (0.06) | |
| T. pseudonana (CCMP1335) | Exponential | 1.25 (0.17) | |
| P. calceolata (RCC100) | Exponential | 1.23 (0.08) | |
| P. calceolata (RCC100) | Stationary | 0.98 (0.11) |
2. Impact of glycerol and Tris buffer concentration on BCA assay, where pct denotes percentage.
3. Comparison of BCA assay response in precipitated protein dissolved in SDS vs sarcosine
4. The comparison involves BSA standard solutions prepared using only sarcosine and 50 mM Tris pH 7 buffer, with incubation times of 30 minutes and 60 minutes. Notably, the removal of glycerol aims to mitigate the BCA assay's sensitivity to incubation duration.
5. For microplate loading:
(1) Reverse pipetting: aspire 200 uL sample from the middle of the solution
(2) Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
(3) Reverse pipet another 200 uL as replicate
(4) Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
Protocol materials
N-lauroylsacosine sodium salt solution (30%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #61747
Step 6
Pierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
In 3 steps
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)Thermo ScientificCatalog #Thermo Scientific™ 0023210
In 3 steps
Sodium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L3771
Step 4.2
Chloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
Step 87
Methanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34860-4X2L-R
Step 88
GlycerolBioshopCatalog #GLY001.500
Step 4.1
EDTA buffer solution (0.5 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #4055-100ml
Step 4.3
Tris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500
In 2 steps
Safety warnings
Waste from BCA assay needs to be collected into waste container and gets further treated before disposal due to the negative impact on the activity of microorganism.
Solvent from protein precipitation (1) the mixture of water and methanol (2) the mixture of methanol and chloroform shall be disposed into separated waste container.
Sample collection
Sample collection
Microalgae samples
Calculate the volume to obtain enough biomass for the assay:
If using 500 uL extraction buffer, the minimum sampling volume (mL) = 750/(Chl-a_ug/L)
If using 1000 uL extraction buffer, the minimum sampling volume (mL) = 2X750/(Chl-a_ug/L)
Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg).
Rinse filter tunnel with filtered artificial seawater (nutrient free) to avoid sample loss.
Fold the filter with two tweezers:
(1) Fold in half along its diameter, creating a semicircular shape;
(2) Fold once more in the same direction, resulting in a long strip;
(3) Fold once more, halving its length, so that sample is secured.
Place folded filter in 2 mL cryogenic vial.
Filter blank media (without cells) through polycarbonate filter as blank.
Flash-freeze tubes with liquid nitrogen and store at -80 °C
Freeze-dry samples before extraction.
Zooplankton samples
Grind freeze-dried samples in metal grinding tube (need dry ice)
Equipment
Metal lysing matrix tube
NAME
MPBio
BRAND
116992006
SKU
Equipment
CoolPrep™ adapter for 24 x 2 mL tube holder on FastPrep-24
NAME
MPBio
BRAND
116002528
SKU
Equipment
FastPrep-24 5G
NAME
Bead-beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
5s
Transfer ground sample into Lysing matrix tube (containing 1.4 mm ceramic spheres, 0.1 mm silica spheres and one 4 mm glass bead), weigh the biomass and log into sampling sheet.
Equipment
Lysing matrix E tube
NAME
2 mL
TYPE
MPbio
BRAND
116914050-CF
SKU
Equipment
Prefilled tubes
NAME
100 µm and 4 mm silica and 1.4 mm zirconia acid washed beads
TYPE
Cole-Parmer
BRAND
2303-MM3
SKU
Flash-freeze tubes with liquid nitrogen, store at -80 °C .
Reagent
Reagent
100 mM pH 7 Tris buffer
Dilute 1 part Tris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500 with 9 part MilliQ.
Protein extraction buffer (PEB, 4X)
Use a transfer pipet to add 40 g GlycerolVWR InternationalCatalog #GLY001.500 into a 100 mL reagent bottle
Weigh 8 g Sodium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L3771 and transfer to the same reagent bottle
Add 4 mL EDTA buffer solution (0.5 M)VWR InternationalCatalog #4055-100ml
Top to 100 g with 100 mM pH 7 Tris buffer.
Shake at 200 rpm, 37°C for complete dissolution.
Note
PEB (4X) might precipitate at room temperature due to the high concentration of glycerol and SDS. Redissolve by shaking at 37 degree.
50 mM pH 7 Tris buffer
Dilute five part Tris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500 with 95 part MilliQ.
20% Sarcosine
Dilute two part N-lauroylsacosine sodium salt solution (30%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #61747 with one part 50 mM pH 7 Tris buffer
Protein extraction buffer (PEB, 1X): 2% SDS, 10% glycerol, 5 mM EDTA in 100 mM pH 7 Tris buffer:
one part Protein extraction buffer (PEB, 4X)
three part 100 mM pH 7 tris buffer
Protein extraction
Protein extraction
Remove samples out of the ULT and keep them On ice .
Prepare samples for homogenizing
Zooplankton samples are already in the bead tubes.
Phytoplankton samples need to be transferred from cryogenic tubes to bead tubes by following the steps below:
Rinse forceps with 95% ethanol and air dry
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Label Lysing matrix tubes (containing 1.4 mm ceramic spheres, 0.1 mm silica spheres and one 4 mm glass bead) and use clean forceps to transfer samples and blank filters into its corresponding tube.
Prior to transferring:
(1) For filters folded into half-strip, unfold once to return to a strip.
(2) For filters folded into quarter-circles, unfold once to return to a half-circle shape, then fold once along the dimension to form a strip.
(2) For filters haphazardly into a compact mass, carefully unfold with two tweezers (avoiding losing biomass), fold once into a half-cricle shape, then fold once more along the dimension to form a strip
Insert the strip into beads mixture.
Place the bead tubes On ice .
Reverse pipet 1 mL PEB (1X) into each bead tube.
Turn on FastPrep
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
Turn on the refrigerate centrifuge, set temperature at 4 °C
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU
Check the cap of each tube to ensure cap is tightly closed. Organize the tubes in order, log the position of each tube, in case the labels get rubbed out during extraction.
Run 00:01:00 at 6.5 m/s
1m
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
1m
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
1m
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
1m
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
Keep tubes On ice for 00:01:00
Check labels. Put tubes back into FastPrep.
Run 00:01:00 at 6.5 m/s
De-foam by centrifuging the extract.
20000 rcf, 4°C, 00:05:00
5m
Transfer extract to 2 mL microtubes.
Centrifuging the extract.
20000 rcf, 4°C, 00:05:00
5m
- Transfer 100 uL supernatant to 2 mL microtube for crude protein measurement.
- Transfere 100 uL supernatant to 2 mL microtube in duplicate for protein precipitation.
- Save the remaining extract.
Freeze at -80 °C .
Crude protein quantitation
Crude protein quantitation
Thaw protein extract ( go to step #39 )
Organize eight 2 mL microtubes in the tube rack, label the tubes from CS1 to CS8 (C denotes crude).
Mix well, and then transfer about 800 uL Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)VWR InternationalCatalog #Thermo Scientific™ 0023210 from the original package into a 2 mL microtube.
PEB and BSA: transferred by reverse pipetting
| Standard | PEB (4X) uL | 100 mM Tris pH 7 (uL) | BSA (2 mg/mL) (uL) | Final Conc. (mg/mL) | |
| CS1 | 125 | 375 | 0 | 0 | |
| CS2 | 125 | 370 | 5 | 0.02 | |
| CS3 | 125 | 363 | 12 | 0.048 | |
| CS4 | 125 | 350 | 25 | 0.1 | |
| CS5 | 125 | 325 | 50 | 0.2 | |
| CS6 | 125 | 275 | 100 | 0.4 | |
| CS7 | 125 | 175 | 200 | 0.8 | |
| CS8 | 125 | 125 | 250 | 1 |
Reverse pipetting: load 4 µL of each standard solution onto microdrop plate.
Equipment
µDrop™ Plates
NAME
Thermo Scientific
BRAND
N12391
SKU
Read absorbance of eight standard solutions at 205 nm
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Subtract absorbance at 205 nm of blank standard from the 205 nm measurements of all other standard solutions
Plot the blank-corrected 205 nm measurement for each standard solution versus its concentration in mg/ml.
Example of BSA standard curve: Absorbance read at 205 nm versus concentration (mg/mL)
If the standard curve has good Coefficient of Determination, i.e., R2>0.99, the standard solutions are in good quality; otherwise, prepare a new series of standard solutions until the quality of standard solutions meets the requirement.
Organize eight 2 mL microtubes in the tube rack, label the tubes from CS1 to CS8. Reverse pipet 100 µL standard solution into its corresponding tube.
Use the following formula to determine the total volume of 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 KitVWR InternationalCatalog #23225
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 50 mL
TYPE
Corning®
BRAND
352070
SKU
Turn on incubator and preheat to 37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Using a single pipette tip and employing the reverse pipetting technique, add 800 µL of WR into each tube. Ensure that the tip does not come into contact with the solution to prevent cross-contamination of samples.
Note
Given the sensitivity of the BCA assay to reaction duration, it is advisable to employ reverse pipetting when dispensing the reagent into all tubes. While the BCA reagent is aqueous, this technique allows for quicker and more consistent dispensing, thereby minimizing any discrepancies in reaction duration between standards and samples, especially for crude protein in PEB with glycerol.
Vortex each tube, shake and incubate at 37 °C for 00:30:00 at 200 rpm.
30m
Remove samples from the incubator and centrifuge 13300 rpm, Room temperature, 00:05:00
5m
Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
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
Calculate protein content per filter
Calculate protein content per filter
Subtract the average absorbance at 562 nm of the blank standard replicates from the 562 nm measurements of all other individual standards.
Subtract the average absorbance at 562 nm of the blank sample (filter) replicates from the 562 nm measurements of all other individual samples.
Create a response curve where the corrected absorbance at 562 nm for each BSA standard is plotted against its concentration in mg/mL.
Standard curve
For crude protein, the standard curve is quadratic.
Where is the blank corrected absorbance at 562 nm and is the concentration of protein.
For precipitated protein, the standard curve is linear.
Where is the blank corrected absorbance at 562 nm and is the concentration of protein.
Use the standard curve to determine the protein concentration of each unknown sample by using its corrected absorbance at 562 nm.
Actual detection limit of the assay (
Enter the standard deviation (SD) of the absorbance values from the blank as in the standard curve to determine the .
If the calculated sample concentration is lower than but higher than 5 ug, go to Section:
Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
If the calculated sample concentration is lower than 5 ug, then use "<LOD" as the result, indicating the protein content is undetectable.
Protein_mg/filter = Protein_mg/mL X PEB_mL
Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
Response of absorbance at 562 nm to BSA concentration after 30-min incubation at 37 and 60 ºC
Thaw extract: remove extract ( go to step #39 ) from ULT and centrifuge 13300 rpm, Room temperature, 00:05:00
5m
Reverse pipetting: Transfer 100 µL supernatant to a new 2 mL microtube.
Return the remaining extract to ULT as soon as possible.
Turn on dry bath and preheat to 60 °C
Equipment
Digital dry bath
NAME
LSE
TYPE
Corning
BRAND
6875SB
SKU
Prepare BSA standard: 0.4 mg/mL
Well mix the package and then directly reverse pipet 300 µL BSA standard into a 2 mL microtube (do not return remaining solution back into the bottle).
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)VWR InternationalCatalog #Thermo Scientific™ 0023210
Forward pipet 600 µL + 600 µL 100 mM pH 7 Tris buffer into the tube, vortex.
Organize eight 2 mL microtubes in the tube rack, label the tubes from CS1 to CS8.
PEB and BSA: transferred by reverse pipetting
| Standard | PEB (4X) (uL) | 100 mM Tris pH 7 (uL) | BSA (0.4 mg/mL) (uL) | Final Conc. (mg/mL) | |
| CS1 | 25 | 75 | 0 | 0 | |
| CS2 | 125 | 370 | 5 | 4 | |
| CS3 | 125 | 365 | 10 | 8 | |
| CS4 | 125 | 355 | 20 | 16 | |
| CS5 | 125 | 345 | 30 | 24 | |
| CS6 | 125 | 315 | 60 | 48 | |
| CS7 | 125 | 250 | 125 | 100 | |
| CS8 | 125 | 125 | 250 | 200 |
Prepare seven 2 mL microtubes for CS2 to CS8.
Vortex and then use reverse pipetting: transfer 100 µL standard solutions into the corresponding tubes, except for CS1 (it has already been 100 uL).
Use the following formula to determine the total volume of 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 KitVWR InternationalCatalog #23225
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 50 mL
TYPE
Corning®
BRAND
352070
SKU
Reverse pipetting: Add 800 µL WR into each tube.
Vortex each tube, incubate at 60 °C for 00:30:00
Cool down samples to room temperature, and centrifuge 13300 rpm, Room temperature, 00:05:00
5m
Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
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 refers to the section: Calculate protein content per filter
Protein precipitation
Protein precipitation
50m 30s
Protein extract from go to step #39
Turn on refrigerate centrifuge, set temperature at 4 °C
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU
Add 300 µL MilliQ into each tube with extract.
In the fume hood, vortex after adding 100 µL Chloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
In the fume hood, add 400 µL Methanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34860-4X2L-R
Vortex until the mixture turns milky.
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
Incubate in the fridge for 00:30:00
30m
20000 rcf, 4°C, 00:10:00
10m
In the fume hood, remove upper phase (about 700 uL)
Note
Do not disturb the interphase.
In the fume hood, add 300 µL methanol. Invert the tube gently at a slight angle to avoid letting precipitated protein and liquid touch the inside of the cap.
20000 rcf, 4°C, 00:10:00
10m
In the fume hood, remove all solvent by using 100 uL pipet tip.
Note
Watch the pipet tip closely. Do not remove protein pellets with the solvent.
With microtube lid open and vacuum pump running, dry pellet in vacuum desiccator for 00:15:00 at Room temperature
Note
Methanol and chloroform interfere the BCA assay. However, do not dry protein pellet for too long, otherwise it might lead to difficulties in resolubilizing the pellet .
15m
Store at -80 °C .
BCA assay to quantify precipitated protein
BCA assay to quantify precipitated protein
50m 30s
Re-solubilization buffer (RSB):
Add 500 µL 20% sarcosine (go to step #6 ) into 9.5 mL 50 mM Tris (go to step #5 )
Reverse pipetting, add 100 µL RSB (go to step #99 ) to each tube with precipitated protein (go to step #98 ).
Vortex for complete re-solubilization.
Transfer about 800 uL Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)VWR InternationalCatalog #Thermo Scientific™ 0023210
to a 2 mL microtube.
Organize eight 2 mL microtubes in the tube rack, label the tubes from PS1 to PS8, P denotes precipitated.
BSA standard solutions
| Standards | 20% sarcosine (uL) | 50 mM Tris pH 7 (uL) | 2 mg/mL BSA (uL) | Final Conc. (mg/mL) | |
| PS2 | 25 | 470 | 5 | 0.02 | |
| PS3 | 25 | 463 | 12 | 0.048 | |
| PS4 | 25 | 450 | 25 | 0.1 | |
| PS5 | 25 | 425 | 50 | 0.2 | |
| PS6 | 25 | 375 | 100 | 0.4 | |
| PS7 | 25 | 275 | 200 | 0.8 | |
| PS8 | 25 | 225 | 250 | 1 |
Reverse pipet 100 µL RSB (go to step #99 ) into PS1.
Prepare seven 2 mL microtubes for PS2 to PC8.
Vortex and then use reverse pipetting: transfer 100 µL standard solutions (PS2 to PS8) into the new tubes.
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 KitVWR InternationalCatalog #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.
Vortex each tube, shake and incubate at 37 °C for 00:30:00 at 200 rpm.
30m
Remove samples from the incubator, and centrifuge 13300 rpm, Room temperature, 00:05:00
5m
Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
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 refers to the section: Calculate protein content per filter