Total crude protein in plankton: Pierce BCA protein assay (including the enhanced assay for low biomass)

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

Value	Label
Metal lysing matrix tube	NAME
MPBio	BRAND
116992006	SKU
www.mpbio.com	LINK

Equipment

Value	Label
CoolPrep™ adapter for 24 x 2 mL tube holder on FastPrep-24	NAME
MPBio	BRAND
116002528	SKU
www.mpbio.com	LINK

Equipment

Value	Label
FastPrep-24 5G	NAME
Bead-beater	TYPE
MP Biomedicals	BRAND
116005500	SKU

Transfer ground sample into Lysing matrix tube, weigh the biomass and log into sampling sheet.

Equipment

Value	Label
Lysing matrix tube I	NAME
MPBio	BRAND
116918100	SKU
www.mpbio.com	LINK

Flash-freeze tubes with liquid nitrogen, store at -80°C until further processing

Freeze dry samples before processed.

Bead size and lysing cycles have impact on protein extraction efficiency.

Bead size

A	B	C	D
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.

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 10g PSB

Use anti-statics weighing dish to weigh the following chemicals (one chemical one dish):

Equipment

Value	Label
Antistatic weighing dish	NAME
Fisherbrand	BRAND
08-732-112	SKU
https://www.fishersci.com/us/en/home.html	LINK

(1) 0.136g Tris base

(2) 0.133g Tris HCl

(3) 0.8g Lithium dodecyl sulphate

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

Value	Label
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 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 4g glycerol into the tube

Add 40µL 0.5Molarity (M) EDTA into the tube

Top to 10g with MilliQ water

Vortex until all solutes are completely dissolved.

Note

Pefabloc is a protease inhibitor, and it loses activity over 24 hours.

Add 20.86mL MilliQ into 100mg Pefabloc to obtain a final concentration of 20millimolar (mM).

Aliquot into 2.5 mL portions and keep frozen at -20°C

The solution can be frozen~thawed multiple times.

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

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

Label bead tubes and use clean forceps to transfer samples and blank filters into its corresponding bead tube.

Reverse pipet 1mL 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.

Turn on FastPrep

Equipment

Value	Label
FastPrep-24 5G	NAME
Bead beater	TYPE
MP Biomedicals	BRAND
116005500	SKU

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 0h 1m 0s at 6.5 m/s

Keep tubes On ice for 0h 1m 0s

Check labels. Put tubes back into FastPrep.

Run 0h 1m 0s at 6.5 m/s

Keep tubes On ice for 0h 1m 0s

Check labels. Put tubes back into FastPrep.

Run 0h 1m 0s at 6.5 m/s

Keep tubes On ice for 0h 1m 0s

Check labels. Put tubes back into FastPrep.

Run 0h 1m 0s at 6.5 m/s

Keep tubes On ice for 0h 1m 0s

De-foam by centrifuging the extract.

2 mL Lysing Matrix tubes at 13000rpm,4Room temperature

15 mL Teenprep tube at 3200x g,4Room temperature

Transfer extract

Microalgae samples in QuickPrep tube (2 mL)

(1) Transfer all supernatant to a 2 mL microtube

(2) Centrifuge at 13000rpm,4Room temperature to spin down debris

(3) Transfer only clear supernatant to a new microtube.

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 13000rpm,4Room temperature to spin down debris

(3) Transfer only clear supernatant to a new microtube.

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 13000rpm,4Room temperature to completely remove debris, and keep the filtrate, discard the filter tube.

Equipment

Value	Label
Costar® Spin-X® Centrifuge Tube Filters, Corning®	BRAND
33500-692	SKU

Equipment

Value	Label
Microcentrifuge tube	NAME
Corning	BRAND
29442-590	SKU

Freeze at -80°C

Thaw 20millimolar (mM) pefabloc and transfer 150 ul to a 600 ul microtube.

Thaw extract in the fridge.

Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.

Reverse pipetting: dispense 125µL PSB into each microtube.

Reverse pipetting: dispense 10µL pefabloc into each microtube

Forward pipetting: Add MilliQ into each microtube according to the sheet below:

A	B	C	D	E	F
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.

If BSA (2 mg/mL) is in ampule, break the ampule with ample opener.

Equipment

Value	Label
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

Vortex each tube.

Reverse pipetting: load 4µL of each standard solution onto microdrop plate.

Equipment

Value	Label
µDrop™ Plates	NAME
Thermo Scientific	BRAND
N12391	SKU
https://www.lifetechnologies.com	LINK

Read absorbance of eight standard solutions at 205 nm

Equipment

Value	Label
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.

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 100µL 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 + # blank filters) X (800µL) = 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

Equipment

Value	Label
Falcon® Centrifuge Tubes	NAME
Polypropylene, Sterile, 50 mL	TYPE
Corning®	BRAND
352070	SKU

Turn on incubator and preheat to 37°C

Equipment

Value	Label
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 100µL extract of blanks or samples into the corresponding tubes.

Use one tip and reverse pipetting: Add 800µL 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 0h 30m 0s

Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate

Equipment

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

Remove samples from the incubator and centrifuge 13300rpm

For microplate loading:

Shake for 5 s at 600 rpm in a continuous and high force modeRead endpoint 562 nm with a measurement time 100 ms

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

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

Thaw 20millimolar (mM) pefabloc and transfer 150 ul to a 600 ul mcirotube.

Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.

Reverse pipetting: dispense 125µL PSB into each microtube.

Reverse pipetting: dispense 10µL pefabloc into each microtube

Forward pipetting: Add MilliQ into each microtube according to the sheet below:

A	B	C	D	E	F
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: 0.4mg/mL

If BSA (2 mg/mL) is in 50 mL bottle, directly reverse pipet 300µL BSA standard into a 2 mL microtube (do not return remaining solution back into the bottle). Forward pipet 600µL+ 600µL Milli-Q into the tube, vortex.

If BSA (2 mg/mL) is in ampule, break the ampule with ample opener. Reverse pipet 300µL BSA standard into a 2 mL microtube. Forward pipet 600µL+ 600µL Milli-Q into the tube,

Equipment

Value	Label
SCIENCEWARE® Break-Safe™ Ampule Opener	NAME
Bel-Art®	BRAND
89217-378	SKU

Reverse pipet certain amount of BSA (0.4mg/ml) into each tube according to the sheet

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 100µL 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 (800µL) = 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

Equipment

Value	Label
Falcon® Centrifuge Tubes	NAME
Polypropylene, Sterile, 50 mL	TYPE
Corning®	BRAND
352070	SKU

Turn on dry bath and preheat to 60°C

Equipment

Value	Label
Digital dry bath	NAME
LSE	TYPE
Corning	BRAND
6875SB	SKU
https://www.fishersci.com/us/en/home.html	LINK

Keep extracted samples On ice

Organize 2 mL microtubes in the tube rack, label the tubes for blanks and samples

Forward pipetting: Add 800µL WR into the tubes.

Reverse pipetting: transfer 100 µL extract of blanks or samples into the corresponding tubes.

Vortex each tube, incubate at 60°C for 0h 30m 0s

Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate

Equipment

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

For microplate loading:

Shake for 5 s at 600 rpm in a continuous and high force modeRead endpoint 562 nm with a measurement time 100 ms

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU