Dec 12, 2022
Version 3
Total crude protein in plankton: Pierce BCA protein assay (including the enhanced assay for low biomass) V.3
- Ying-Yu Hu1,
- Christopher Lord1,
- Zoe V. Finkel1
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu, Christopher Lord, Zoe V. Finkel 2022. Total crude protein in plankton: Pierce BCA protein assay (including the enhanced assay for low biomass) . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoy5e7g4o/v3Version created by Ying-Yu Hu
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 13, 2022
Last Modified: December 12, 2022
Protocol Integer ID: 69957
Keywords: microalgae, total protein, Pierce BCA, protein solubilization buffer, bead mill cell disruption, microplate, zooplankton
Funders Acknowledgement:
Simons Collaborative on Ocean Processes and Ecology
Grant ID: 723789
Simons Collaboration on Computational Biogeochemical Modeling of Marine Ecosystems
Grant ID: 549937
Abstract
Here we describe a protocol for extracting total crude protein from phytoplankton and zooplankton, and quantifying by Pierce BCA protein assay. Chlorophyll, phospholipids and sucrose in crude protein could interfere the BCA assay.
Guidelines
- Working range of Pierce BCA assay is 20-2000 ug/ml protein.
- Enhanced assay has working range of 5 to 200 ug/mL protein.
- Minimum sampling volume for microalgae (mL) = 750/(Chl-a_ug/L), if protein is extracted by only 500 ul extraction buffer instead of 1 mL.
- One extra step of using microspin centrifuge filter is required for protein from zooplankton or microalgae samples with fine debris .
Protocol materials
Tris baseBioshopCatalog #TRS001.500
Step 7.1
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)Thermo ScientificCatalog #Thermo Scientific™ 0023210
In 2 steps
Pierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
In 2 steps
BSA 2 mg/mL standardThermo Fisher ScientificCatalog #23209
In 2 steps
Tris HClBioshopCatalog #TRS002.500
Step 7.1
Lithium Dodecyl SulphateBioshopCatalog #LDS701.25
Step 7.1
GlycerolBioshopCatalog #GLY001.500
Step 7.6
EDTA buffer solution (0.5 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #4055-100ml
Step 7.7
4-(2-2-aminoethyl)-benzenesulfonyl fluoride HCL (AEBSF, Pefabloc)BioshopCatalog #AEB602.100
Step 8
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.
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.
Place sample filters in 2 mL Cryogenic Vials.
Filter blank media (without cells) through polycarbonate filter as blank.
Flash-freeze tubes with liquid nitrogen and store at -80 °C
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
Transfer ground sample into Lysing matrix tube, weigh the biomass and log into sampling sheet.
Equipment
Lysing matrix tube I
NAME
MPBio
BRAND
116918100
SKU
Flash-freeze tubes with liquid nitrogen, store at -80 °C until further processing
Freeze dry samples before processed.
Bead tube test for Microalgae samples
Bead tube test for Microalgae samples
Bead size and lysing cycles have impact on protein extraction efficiency.
Bead size
Note
- Use 2 ml lysing matrix tube for 25 mm filter; 15 ml Teenprep tube for 47 mm filter
| Lysing tube | Bead size (mm) | Composition | 2 mL tube SKU | |
| Matrix B | 0.1 | Silica spheres | 116911050-CF | |
| Matrix Y | 0.5 | Yttria-stabilized zirconium oxide beads | 116960050-CF | |
| Matrix C | 1 | Silica spheres | 116912050-CF | |
| Matrix D | 1.4 | Zirconium-Silica spheres | 116913050-CF |
Lysing cycles
Compare protein yield by using four, six and eight cycles
Use the optimized bead size and lysing cycles to process protein samples.
Prepare protein solubilization buffer (PSB)
Prepare protein solubilization buffer (PSB)
CITATION
In order to obtain compatible results, prepare sufficient PSB so that the same PSB can be used for sample extraction, blank filter extraction and standard solutions
(1) Extract all samples: Each sample requires 0.25 mL PSB
(2) Extract all blank filters: Each filter requires 0.25 mL PSB
(3) Each standard solution (500 ul) requires 0.125 mL PSB
For each 10 g PSB
Use anti-statics weighing dish to weigh the following chemicals (one chemical one dish):
Equipment
Antistatic weighing dish
NAME
Fisherbrand
BRAND
08-732-112
SKU
(1) 0.136 g Tris base
Tris baseVWR InternationalCatalog #TRS001.500
(2) 0.133 g Tris HCl
Tris HClVWR InternationalCatalog #TRS002.500
(3) 0.8 g Lithium dodecyl sulphate
Lithium Dodecyl SulphateVWR InternationalCatalog #LDS701.25
Place a plastic beaker on the top of the scale surface
Remove the cap of a 15 mL tube and sit it in the beaker
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 15 mL
TYPE
Corning®
BRAND
352096
SKU
Tare the total weight of beaker and tube
Transfer all chemicals weighed in go to step #7.1 into the tube, rinse the dish with small amount of MilliQ water to make certain all of the solutes is transferred into the tube
Use a transfer pipet to add 4 g glycerol into the tube
GlycerolVWR InternationalCatalog #GLY001.500
Add 40 µL 0.5 Molarity (M) EDTA into the tube
EDTA buffer solution (0.5 M)VWR InternationalCatalog #4055-100ml
Top to 10 g with MilliQ water
Vortex until all solutes are completely dissolved.
Note
It takes some time to have solutes dissolved, especially LDS. Prepare in advance.
Prepare Pefabloc solution
Prepare Pefabloc solution
4-(2-2-aminoethyl)-benzenesulfonyl fluoride HCL (AEBSF, Pefabloc)VWR InternationalCatalog #AEB602.100
Note
Pefabloc is a protease inhibitor, and it loses activity over 24 hours.
Add 20.86 mL MilliQ into 100 mg Pefabloc to obtain a final concentration of 20 millimolar (mM) .
Aliquot into 2.5 mL portions and keep frozen at -20 °C
The solution can be frozen~thawed multiple times.
Assay Day 1: Extract protein
Assay Day 1: Extract protein
Prepare protein extraction buffer (PEB):
Each 1 mL PEB contains
250 ul PSB
20 ul 20 mM Pefabloc
730 ul MilliQ water
Prepare ice-bath, keep all samples in the ice-bath
Rinse forceps with 70% ethanol and air dry
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Label bead tubes and use clean forceps to transfer samples and blank filters into its corresponding bead tube.
Note
Bead tube type is selected by go to step #4
Reverse pipet 1 mL PEB onto the filter.
When using 15 mL Teenprep tube, horizontally shake the tube to bury filter into beads before adding PEB, which makes filter easy to be homogenized.
Note
Volume of PEB varies due to the actual biomass collected (see guideline)
Turn on FastPrep
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
Check the cap of each tube to make certain cap is tightly screwed. Organize the tubes in order, take notes of 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
Note
More cycles might be requiredgo to step #4
De-foam by centrifuging the extract.
2 mL Lysing Matrix tubes at 13000 rpm, Room temperature, 00:05:00
15 mL Teenprep tube at 3200 x g, Room temperature, 00:05:00
10m
Transfer extract
Microalgae samples in QuickPrep tube (2 mL)
(1) Transfer all supernatant to a 2 mL microtube
(2) Centrifuge at 13000 rpm, Room temperature, 00:05:00 to spin down debris
(3) Transfer only clear supernatant to a new microtube.
5m
Microalgae samples in TeenPrep tube (15 mL)
(1) Use 200 uL tip, go straight to the bottom along the side, try to transfer all extract to a 2 mL microtube.
(2) Centrifuge at 13000 rpm, Room temperature, 00:05:00 to spin down debris
(3) Transfer only clear supernatant to a new microtube.
5m
Zooplankton samples and Microalgae samples with cloudy extract in which debris is too fine to be centrifuged down
(1) Use puncher to cut glass fibre filters into about 7 mm disks
(2) Insert centrifuge filter tube into 2 mL microtube, line the bottom with two glass fibre filter disks by using ethanol rinsed and air-dried tweezers
(3) Transfer all extract to filter tube
(4) Centrifuge at 13000 rpm, Room temperature, 00:05:00 to completely remove debris, and keep the filtrate, discard the filter tube.
Equipment
new equipment
NAME
Costar® Spin-X® Centrifuge Tube Filters, Corning®
BRAND
33500-692
SKU
Equipment
Microcentrifuge tube
NAME
Corning
BRAND
29442-590
SKU
5m
Freeze at -80 °C
Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
Thaw 20 millimolar (mM) pefabloc and transfer 150 ul to a 600 ul microtube.
Note
Put the rest of the stock back into the freezer immediately.
Thaw extract in the fridge.
Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.
Reverse pipetting: dispense 125 µL PSB into each microtube.
Note
When aspiring solution, ensure the pipette to be held vertically. When dispensing, ensure you hold the pipette at an angle (10-45°). Working to these angles ensures the desired liquid amount is drawn into the tip properly and that all of the liquid is fully dispensed without leaving any residue in the tip.
Reverse pipetting: dispense 10 µL pefabloc into each microtube
Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
Forward pipetting: Add MilliQ into each microtube according to the sheet below:
| Standard | PSB (uL) | Pefabloc (uL) | MQ (uL) | BSA (2 mg/mL) (uL) | Final Conc. (mg/mL) | |
| SD1 | 125 | 10 | 365 | 0 | 0 | |
| SD2 | 125 | 10 | 360 | 5 | 0.02 | |
| SD3 | 125 | 10 | 353 | 12 | 0.048 | |
| SD4 | 125 | 10 | 340 | 25 | 0.1 | |
| SD5 | 125 | 10 | 315 | 50 | 0.2 | |
| SD6 | 125 | 10 | 265 | 100 | 0.4 | |
| SD7 | 125 | 10 | 165 | 200 | 0.8 | |
| SD8 | 125 | 10 | 115 | 250 | 1 |
Primary BSA standard
If BSA (2 mg/mL) is in 50 mL bottle, transfer 1 mL into a microtube.
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)VWR InternationalCatalog #Thermo Scientific™ 0023210
If BSA (2 mg/mL) is in ampule, break the ampule with ample opener.
BSA 2 mg/mL standardVWR InternationalCatalog #23209
Equipment
SCIENCEWARE® Break-Safe™ Ampule Opener
NAME
Bel-Art®
BRAND
89217-378
SKU
Reverse pipet certain amount of BSA (2 mg/mL) into each tube according to the sheet go to step #39
Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
Vortex each tube.
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.
Standard solutions can be kept at Room temperature .
Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8. Reverse pipet 100 µL standard solution into its corresponding tube.
Assay Day 2: Prepare BCA working reagent (WR)
Assay Day 2: Prepare BCA working reagent (WR)
Use the following formula to determine the total volume of WR required. Consider leaving several mL of extra volume:
(# standards + # samples + # blank filters) 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
Assay Day 2: Pierce BCA assay
Assay Day 2: Pierce BCA assay
2h
2h
Turn on incubator and preheat to 37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Keep thawed extract On ice
Organize 2 mL microtubes in the tube rack, label the tubes for blanks and samples
Vortex and then use reverse pipetting: transfer 100 µL extract of blanks or samples into the corresponding tubes.
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 reaction 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
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
Example of organizing samples on the microplate.
Remove samples from the incubator and centrifuge 13300 rpm, Room temperature, 00:05:00
5m
For microplate loading:
Note
- Reverse pipetting: aspire 200 µL sample from the middle of the solution
- Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
- Dispose the tip
- Use a new tip, reverse pipet another 200 µL as replicate
- Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
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 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.
Use the standard curve to determine the protein concentration of each unknown sample by using its blank-corrected 562 absorbance.
Calculate the low-limit-of-detection:
L-LOD_mg/mL=3.3*SD/slope
where SD is the mean value of standard deviation between each standard replicates.
L-Abs=L-LOD*slope - intercept
If the absorbance of sample is lower than L-Abs, go to Section:
Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
Protein_mg/filter = Protein_mg/mL X PEB_mL
Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
Assay Day 3: 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 20 millimolar (mM) pefabloc and transfer 150 ul to a 600 ul mcirotube.
Note
Put the rest of the stock back into the freezer immediately.
Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.
Reverse pipetting: dispense 125 µL PSB into each microtube.
Note
When aspiring solution, hold the pipet vertically. When dispensing, ensure you hold the pipette at an angle (10-45°). Working to these angles ensures the desired liquid amount is drawn into the tip properly and that all of the liquid is fully dispensed without leaving any residue in the tip.
Reverse pipetting: dispense 10 µL pefabloc into each microtube
Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
Forward pipetting: Add MilliQ into each microtube according to the sheet below:
| Standard | PSB (uL) | Pefabloc (uL) | MQ (uL) | BSA (0.4 mg/mL) (uL) | Final Conc. (mg/mL) | |
| SD1 | 125 | 10 | 365 | 0 | 0 | |
| SD2 | 125 | 10 | 360 | 5 | 4 | |
| SD3 | 125 | 10 | 355 | 10 | 8 | |
| SD4 | 125 | 10 | 345 | 20 | 16 | |
| SD5 | 125 | 10 | 335 | 30 | 24 | |
| SD6 | 125 | 10 | 305 | 60 | 48 | |
| SD7 | 125 | 10 | 240 | 125 | 100 | |
| SD8 | 125 | 10 | 115 | 250 | 200 |
Prepare BSA standard: 0.4 mg/mL
If BSA (2 mg/mL) is in 50 mL bottle, directly reverse pipet 300 µL BSA standard into a 2 mL microtube (do not return remaining solution back into the bottle). Forward pipet 600 µL + 600 µL Milli-Q into the tube, vortex.
Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard 2 mg/mL (50 mL)VWR InternationalCatalog #Thermo Scientific™ 0023210
If BSA (2 mg/mL) is in ampule, break the ampule with ample opener. Reverse pipet 300 µL BSA standard into a 2 mL microtube. Forward pipet 600 µL + 600 µL Milli-Q into the tube,
BSA 2 mg/mL standardVWR InternationalCatalog #23209
Equipment
SCIENCEWARE® Break-Safe™ Ampule Opener
NAME
Bel-Art®
BRAND
89217-378
SKU
Reverse pipet certain amount of BSA (0.4 mg/ml ) into each tube according to the sheet go to step #75
Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
Vortex each tube.
Standard solutions can be kept at Room temperature .
Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8. 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 since Finntip stepper is unable to expel the entire volume from the tip:
(# standards + # samples + # blank filters) 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 dry bath and preheat to 60 °C
Equipment
Digital dry bath
NAME
LSE
TYPE
Corning
BRAND
6875SB
SKU
Keep extracted samples On ice
Organize 2 mL microtubes in the tube rack, label the tubes for blanks and samples
Forward pipetting: Add 800 µL WR into the tubes.
Reverse pipetting: transfer 100 µL extract of blanks or samples into the corresponding tubes.
Note
Aspire and mix up and down at least three time before transferring the extract
Vortex each tube, incubate at 60 °C for 00:30:00
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
Example of organizing samples on the microplate.
For microplate loading:
Note
- Reverse pipetting: aspire 200 µL sample
- Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
- Return remaining sample from the tip back to the tube
- Dispose the tip
- Vortex the tube
- Use a new tip, reverse pipet another 200 µL as replicate
- Tip gently touches the side of the well, avoid bending. Dispense 200 uL into the microplate
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
Citations
Step 5
Ni G, Zimbalatti G, Murphy CD, Barnett AB, Arsenault CM, Li G, Cockshutt AM, Campbell DA. Arctic Micromonas uses protein pools and non-photochemical quenching to cope with temperature restrictions on Photosystem II protein turnover.
https://doi.org/10.1007/s11120-016-0310-6