TITLE: Total crude protein in plankton: Pierce BCA protein assay (including the enhanced assay for low biomass) 
AUTHORS: Ying-Yu Hu, Christopher Lord, Zoe V. Finkel

[DESCRIPTION]
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. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf

[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 .

[STEPS]
SECTION: Sample collection
1. Microalgae samples
SECTION: Sample collection
1.2. Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg). 
SECTION: Sample collection
1.4. Place sample filters in 2 mL Cryogenic Vials.
SECTION: Sample collection
3. Freeze dry samples before processed. 
SECTION: Prepare protein solubilization buffer (PSB)
5. 
 

SECTION: Prepare protein solubilization buffer (PSB)
6. 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
SECTION: Prepare protein solubilization buffer (PSB)
7. For each 10 g PSB

SECTION: Prepare protein solubilization buffer (PSB)
7.1. Use anti-statics weighing dish to weigh the following chemicals (one chemical one dish):

 


(1) 0.136 g Tris base

 

(2) 0.133 g Tris HCl

 

(3) 0.8 g Lithium dodecyl sulphate
 

SECTION: Prepare protein solubilization buffer (PSB)
7.2. Place a plastic beaker on the top of the scale surface

SECTION: Prepare protein solubilization buffer (PSB)
7.3. Remove the cap of a 15 mL tube and sit it in the beaker
 

SECTION: Prepare protein solubilization buffer (PSB)
7.4. Tare the total weight of beaker and tube
SECTION: Prepare protein solubilization buffer (PSB)
7.5. 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

SECTION: Prepare protein solubilization buffer (PSB)
7.6. Use a transfer pipet to add 4 g glycerol into the tube

 

SECTION: Prepare protein solubilization buffer (PSB)
7.7. Add 40 µL 0.5 Molarity (M) EDTA into the tube
 

SECTION: Prepare protein solubilization buffer (PSB)
7.8. Top to 10 g with MilliQ water

SECTION: Prepare protein solubilization buffer (PSB)
7.9. Vortex until all solutes are completely dissolved. 
 

SECTION: Prepare Pefabloc solution
8. 
 

 

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

SECTION: Prepare Pefabloc solution
10. Aliquot into 2.5 mL portions and keep frozen at -20 °C 

SECTION: Prepare Pefabloc solution
11. The solution can be frozen~thawed multiple times.
SECTION: Assay Day 1: Extract protein
12. Prepare protein extraction buffer (PEB):

Each 1 mL PEB contains

250 ul PSB
20 ul 20 mM Pefabloc
730 ul MilliQ water
SECTION: Assay Day 1: Extract protein
13. Prepare ice-bath, keep all samples in the ice-bath
SECTION: Assay Day 1: Extract protein
17. Turn on FastPrep 
 

SECTION: Assay Day 1: Extract protein
18. 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. 
SECTION: Assay Day 1: Extract protein
19. Run 1 min at 6.5 m/s

SECTION: Assay Day 1: Extract protein
20. Keep tubes on ice for 1 min 

SECTION: Assay Day 1: Extract protein
21. Check labels. Put tubes back into FastPrep.
SECTION: Assay Day 1: Extract protein
22.  Run 1 min at 6.5 m/s

SECTION: Assay Day 1: Extract protein
23. Keep tubes on ice for 1 min 
SECTION: Assay Day 1: Extract protein
24. Check labels. Put tubes back into FastPrep.
SECTION: Assay Day 1: Extract protein
25. Run 1 min at 6.5 m/s
SECTION: Assay Day 1: Extract protein
26. Keep tubes  on ice for 1 min 
SECTION: Assay Day 1: Extract protein
27. Check labels. Put tubes back into FastPrep.
SECTION: Assay Day 1: Extract protein
28. Run 1 min at 6.5 m/s
SECTION: Assay Day 1: Extract protein
29. Keep tubes on ice for 1 min 
SECTION: Assay Day 1: Extract protein
31. De-foam by centrifuging the extract.
2 mL Lysing Matrix tubes at 13000 rpm, 5 min, 4 Room temperature 
15 mL Teenprep tube at 3200 x g, 5 min, 4 Room temperature 

SECTION: Assay Day 1: Extract protein
32. Transfer extract
SECTION: Assay Day 1: Extract protein
33. Freeze at -80 °C 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
34. Thaw 20 millimolar (mM) pefabloc and transfer 150 ul to a 600 ul microtube. 
 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
36. Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
37. Reverse pipetting: dispense 125 µL PSB into each microtube.
 

  

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
38. Reverse pipetting: dispense 10 µL pefabloc into each microtube
 


SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
39. Forward pipetting: Add MilliQ into each microtube according to the sheet below:
      Standard      PI (20 mM) (µL)      4XPSB (µL)      MQ (uL)      BSA (2mg/mL) (uL)      Total (uL)      Final Conc. (mg/mL)        SD1      10      125      365      0      500      0        SD2      10      125      360      5      500      0.02        SD3      10      125      353      12      500      0.048        SD4      10      125      340      25      500      0.1        SD5      10      125      315      50      500      0.2        SD6      10      125      265      100      500      0.4        SD7      10      125      165      200      500      0.8        SD8      10      125      115      250      500      1   
 
 
 
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
40. Primary BSA standard

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
41. Reverse pipet certain amount of BSA (2 mg/mL) into each tube according to the sheet   

 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
42. Vortex each tube.
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
43. Reverse pipetting: load 4 µL of each standard solution onto microdrop plate.
 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
44. Read absorbance of eight standard solutions at 205 nm
 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
45. Subtract absorbance at 205 nm of blank standard from the 205 nm measurements of all other standard solutions
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
46. Plot the blank-corrected 205 nm measurement for each standard solution versus its concentration in mg/ml.
  

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
47. 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.
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
48. Standard solutions can be kept at Room temperature .
SECTION: Assay Day 2: Prepare BCA working reagent (WR)
50. 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

SECTION: Assay Day 2: Prepare BCA working reagent (WR)
51. Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube

 

 

SECTION: Assay Day 2: Pierce BCA assay
52. Turn on incubator and preheat to 37 °C 
 

SECTION: Assay Day 2: Pierce BCA assay
53. Keep thawed extract on ice 
SECTION: Assay Day 2: Pierce BCA assay
58. Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
 


SECTION: Assay Day 2: Pierce BCA assay
59.   
SECTION: Assay Day 2: Pierce BCA assay
61. For microplate loading:
 

SECTION: Assay Day 2: Pierce BCA assay
62. Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 562 nm with a measurement time 100 ms

 

SECTION: Calculate protein content per filter
63. Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard.
SECTION: Calculate protein content per filter
64. Subtract the average 562 nm absorbance measurement of the blank sample (filter) replicates from the 562 nm measurements of all other individual sample.
SECTION: Calculate protein content per filter
65. Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard versus its concentration in mg/ml.

SECTION: Calculate protein content per filter
66. Use the standard curve to determine the protein concentration of each unknown sample by using its blank-corrected 562 absorbance.
SECTION: Calculate protein content per filter
69. Protein_mg/filter = Protein_mg/mL X PEB_mL
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
49. 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. 

SECTION: Assay Day 2: Pierce BCA assay
54. Organize 2 mL microtubes in the tube rack, label the tubes for blanks and samples
SECTION: Assay Day 2: Pierce BCA assay
55. Vortex and then use reverse pipetting: transfer 100 µL extract of blanks or samples into the corresponding tubes.
SECTION: Assay Day 2: Pierce BCA assay
56. 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. 

 

SECTION: Assay Day 2: Pierce BCA assay
57. Vortex each tube, shake and incubate at 37 °C for 30 min 

SECTION: Calculate protein content per filter
67. 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
SECTION: Calculate protein content per filter
68. 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
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
70. 
  

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
71. Thaw 20 millimolar (mM) pefabloc and transfer 150 ul to a 600 ul mcirotube. 
 


SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
72. Organize eight 2 mL microtubes in the tube rack, label the tubes from SD1 to SD8.
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
73. Reverse pipetting: dispense 125 µL PSB into each microtube.
 

  

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
74. Reverse pipetting: dispense 10 µL pefabloc into each microtube
 

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
75. Forward pipetting: Add MilliQ into each microtube according to the sheet below:
      Standard      PI (20 mM) (µL)      4XPSB (µL)      MQ (uL)      BSA (2mg/mL) (uL)      Total (uL)      Final Conc. (mg/mL)        SD1      10      125      365      0      500      0        SD2      10      125      360      5      500      0.02        SD3      10      125      353      12      500      0.048        SD4      10      125      340      25      500      0.1        SD5      10      125      315      50      500      0.2        SD6      10      125      265      100      500      0.4        SD7      10      125      165      200      500      0.8        SD8      10      125      115      250      500      1   
 
 
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
77. Reverse pipet certain amount of BSA (0.4 mg/ml) into each tube according to the sheet   

 

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
78. Vortex each tube.
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
79. Standard solutions can be kept at Room temperature .
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
80. 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. 

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
81. 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

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
82. Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube

 

 

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
83. Turn on dry bath and preheat to 60 °C 

 



SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
84. Keep extracted samples on ice 
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
85. Organize 2 mL microtubes in the tube rack, label the tubes for blanks and samples
SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
87. Reverse pipetting: transfer 100 µL extract of blanks or samples into the corresponding tubes.
 


SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
86. Forward pipetting: Add 800 µL WR into the tubes.

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
88. Vortex each tube, incubate at 60 °C for 30 min 

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
89. Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
 


SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
90.   

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
91. For microplate loading:
 


SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
92. Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 562 nm with a measurement time 100 ms

 

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
40.1. If BSA (2 mg/mL) is in 50 mL bottle, transfer 1 mL into a microtube. 
  

SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
40.2. If BSA (2 mg/mL) is in ampule, break the ampule with ample opener. 

 

 


SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
76. Prepare BSA standard: 0.4 mg/mL

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
76.1. 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.  
  

SECTION: Assay Day 3: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
76.2. 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, 

 

 


SECTION: Sample collection
1.1. 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)
SECTION: Sample collection
2. Zooplankton samples
SECTION: Sample collection
2.1. Grind freeze-dried samples in metal grinding tube (need dry ice)
 

 

 

SECTION: Sample collection
2.2. Transfer ground sample into Lysing matrix tube, weigh the biomass and log into sampling sheet. 
 

SECTION: Assay Day 1: Extract protein
32.1. Microalgae samples in QuickPrep tube (2 mL)

(1) Transfer all supernatant to a 2 mL microtube
(2) Centrifuge at  13000 rpm, 5 min, 4 Room temperature  to spin down debris
(3) Transfer only clear supernatant to a new microtube. 
SECTION: Assay Day 1: Extract protein
32.3. 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, 5 min, 4 Room temperature  to completely remove debris, and keep the filtrate, discard the filter tube.

 

 

SECTION: Assay Day 1: Extract protein
32.2. 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, 5 min, 4 Room temperature  to spin down debris
(3) Transfer only clear supernatant to a new microtube. 
SECTION: Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions
35. Thaw extract in the fridge.
SECTION: Assay Day 2: Pierce BCA assay
60. Remove samples from the incubator and centrifuge 13300 rpm, 5 min 
SECTION: Bead tube test for Microalgae samples
4. Bead size and lysing cycles have impact on protein extraction efficiency.
SECTION: Bead tube test for Microalgae samples
4.1. Bead size

 

 

SECTION: Bead tube test for Microalgae samples
4.2. Lysing cycles
Compare protein yield by using four, six and eight cycles
SECTION: Bead tube test for Microalgae samples
4.3. Use the optimized bead size and lysing cycles to process protein samples.
SECTION: Assay Day 1: Extract protein
14. Rinse forceps with 70% ethanol and air dry

 

SECTION: Assay Day 1: Extract protein
15. Label bead tubes and use clean forceps to transfer samples and blank filters into its corresponding bead tube.
 

SECTION: Assay Day 1: Extract protein
16. 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.

 

SECTION: Assay Day 1: Extract protein
30. 
 

SECTION: Sample collection
1.3. Rinse filter tunnel with filtered artificial seawater (nutrient free) to avoid sample loss.
SECTION: Sample collection
1.5. Filter blank media (without cells) through polycarbonate filter as blank. 
SECTION: Sample collection
1.6. Flash-freeze tubes with liquid nitrogen and store at -80 °C
SECTION: Sample collection
2.3. Flash-freeze tubes with liquid nitrogen, store at -80 °C until further processing