TITLE: Comprehensive Protocol for Total Protein Extraction, Precipitation, and BCA Assay Measurement from Plankton Samples
AUTHORS: Ying-Yu Hu

[DESCRIPTION]
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.

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

[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.
 
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

[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). 
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1.5. Place folded filter in 2 mL cryogenic vial.
SECTION: Reagent
3. 100 mM pH 7 Tris buffer

Dilute 1 part   with 9 part MilliQ.

SECTION: Protein extraction
9. 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:
SECTION: Protein extraction
8. Remove samples out of the ULT and keep them on ice .

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11. Turn on FastPrep 
 

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13. 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. 
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14. Run 1 min at 6.5 m/s

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15. Keep tubes on ice for 1 min 

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16. Check labels. Put tubes back into FastPrep.
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17.  Run 1 min at 6.5 m/s

SECTION: Protein extraction
18. Keep tubes on ice for 1 min 
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19. Check labels. Put tubes back into FastPrep.
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20. Run 1 min at 6.5 m/s
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21. Keep tubes  on ice for 1 min 
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22. Check labels. Put tubes back into FastPrep.
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23. Run 1 min at 6.5 m/s
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24. Keep tubes on ice for 1 min 
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36. De-foam by centrifuging the extract.
 20000 rcf, 5 min, 4 °C 
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37. Transfer extract to 2 mL microtubes.
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40. Freeze at -80 °C .

SECTION: Crude protein quantitation
42. Organize eight 2 mL microtubes in the tube rack, label the tubes from CS1 to CS8 (C denotes crude).
SECTION: Crude protein quantitation
44. PEB and BSA: transferred by reverse pipetting

      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: Crude protein quantitation
45. Reverse pipetting: load 4 µL of each standard solution onto microdrop plate.
 

SECTION: Crude protein quantitation
46. Read absorbance of eight standard solutions at 205 nm
 

SECTION: Crude protein quantitation
47. Subtract absorbance at 205 nm of blank standard from the 205 nm measurements of all other standard solutions
SECTION: Crude protein quantitation
48. Plot the blank-corrected 205 nm measurement for each standard solution versus its concentration in mg/ml.
 

SECTION: Crude protein quantitation
49. 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: Crude protein quantitation
51. 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
SECTION: Crude protein quantitation
52. Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube

 
 

SECTION: Crude protein quantitation
53. Turn on incubator and preheat to 37 °C 
 

SECTION: Crude protein quantitation
57. Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
 

SECTION: Crude protein quantitation
58. 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
59. Subtract the average absorbance at 562 nm of the blank standard replicates from the 562 nm measurements of all other individual standards.
SECTION: Calculate protein content per filter
60. Subtract the average absorbance at 562 nm of the blank sample (filter) replicates from the 562 nm measurements of all other individual samples.
SECTION: Calculate protein content per filter
61. Create a response curve where the corrected absorbance at 562 nm for each BSA standard is plotted against its concentration in mg/mL.

SECTION: Calculate protein content per filter
63. Use the standard curve to determine the protein concentration of each unknown sample by using its corrected absorbance at 562 nm.
SECTION: Calculate protein content per filter
67. Protein_mg/filter = Protein_mg/mL X PEB_mL
SECTION: Crude protein quantitation
50. 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. 

SECTION: Crude protein quantitation
54. 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.

 

SECTION: Crude protein quantitation
55. Vortex each tube, shake and incubate at 37 °C for 30 min at 200 rpm.

SECTION: Calculate protein content per filter
64. Actual detection limit of the assay (  
SECTION: Calculate protein content per filter
65. 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
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
68. 
 

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
73. Organize eight 2 mL microtubes in the tube rack, label the tubes from CS1 to CS8.
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
74. PEB and BSA: transferred by reverse pipetting
      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: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
75. 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).

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
76. 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
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
77. Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube

 
 

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

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
78. Reverse pipetting: Add 800 µL WR into each tube.
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
79. Vortex each tube, incubate at 60 °C for 30 min 
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
81. Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
 

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
82. 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: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
72. Prepare BSA standard: 0.4 mg/mL
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
72.1. 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). 

  

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
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2.1. Grind freeze-dried samples in metal grinding tube (need dry ice)
 

 

 

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2.2. 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. 
 

 

SECTION: Crude protein quantitation
56. Remove samples from the incubator and centrifuge 13300 rpm, 5 min 
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10. Reverse pipet 1 mL PEB (1X) into each bead tube.
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1.3. Rinse filter tunnel with filtered artificial seawater (nutrient free) to avoid sample loss.
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1.6. Filter blank media (without cells) through polycarbonate filter as blank. 
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1.7. Flash-freeze tubes with liquid nitrogen and store at -80 °C
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2.3. Flash-freeze tubes with liquid nitrogen, store at -80 °C .
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1.4. 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.
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1.8. Freeze-dry samples before extraction.
SECTION: Reagent
4. Protein extraction buffer (PEB, 4X)
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6. 20% Sarcosine
Dilute two part   with one part 50 mM pH 7 Tris buffer
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5. 50 mM  pH 7 Tris buffer

Dilute five part   with 95 part MilliQ.
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7. 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
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25. Check labels. Put tubes back into FastPrep.
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26. Run 1 min at 6.5 m/s
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27. Keep tubes on ice for 1 min 
SECTION: Protein extraction
28. Check labels. Put tubes back into FastPrep.
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29. Run 1 min at 6.5 m/s
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30. Keep tubes on ice for 1 min 
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31. Check labels. Put tubes back into FastPrep.
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32. Run 1 min at 6.5 m/s
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33. Keep tubes on ice for 1 min 
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34. Check labels. Put tubes back into FastPrep.
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35. Run 1 min at 6.5 m/s
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12. Turn on the refrigerate centrifuge, set temperature at 4 °C 
 

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38. Centrifuging the extract.
 20000 rcf, 5 min, 4 °C 
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39. 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. 
SECTION: Crude protein quantitation
43. Mix well, and then transfer about 800 uL   from the original package into a 2 mL microtube.

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
72.2. Forward pipet 600 µL + 600 µL 100 mM pH 7 Tris buffer into the tube, vortex. 
SECTION: Reagent
4.1. Use a transfer pipet to add 40 g   into a 100 mL reagent bottle
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4.2. Weigh 8 g    and transfer to the same reagent bottle
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4.3. Add 4 mL   
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4.4. Top to 100 g with 100 mM  pH 7 Tris buffer.
SECTION: Protein precipitation
84. Protein extract from   

SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
69. Thaw extract: remove extract (  )  from ULT and centrifuge 13300 rpm, 5 min 
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
70. Reverse pipetting: Transfer 100 µL supernatant to a new 2 mL microtube. 
Return the remaining extract to ULT as soon as possible.
SECTION: Protein precipitation
85. Turn on refrigerate centrifuge, set temperature at 4 °C 
 

SECTION: Protein precipitation
86. Add 300 µL  MilliQ into each tube with extract.

SECTION: Protein precipitation
87. In the fume hood, vortex after adding 100 µL   

SECTION: Protein precipitation
88. In the fume hood, add 400 µL   

SECTION: Protein precipitation
89. Vortex until the mixture turns milky.
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90. Gently vortex for 30 s  by using a tube insert
 

SECTION: Protein precipitation
91. Incubate in the fridge for 30 min   
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92.  20000 rcf, 10 min, 4 °C 
SECTION: Protein precipitation
93. In the fume hood, remove upper phase (about 700 uL) 
 

SECTION: Protein precipitation
94. 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.

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95.  20000 rcf, 10 min, 4 °C 
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96. In the fume hood, remove all solvent by using 100 uL pipet tip.
 

SECTION: Protein precipitation
97. With microtube lid open and vacuum pump running, dry pellet in vacuum desiccator for 15 min  at Room temperature   
 

SECTION: BCA assay to quantify precipitated protein
101. Transfer about 800 uL   
to a 2 mL microtube.
SECTION: BCA assay to quantify precipitated protein
103. BSA standard solutions
 


SECTION: BCA assay to quantify precipitated protein
106. Vortex and then use reverse pipetting: transfer 100 µL standard solutions (PS2 to PS8) into the new tubes.
SECTION: BCA assay to quantify precipitated protein
107. 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
SECTION: BCA assay to quantify precipitated protein
108. Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube
  
SECTION: BCA assay to quantify precipitated protein
109. 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: BCA assay to quantify precipitated protein
110. Vortex each tube, shake and incubate at 37 °C for 30 min at 200 rpm.
SECTION: BCA assay to quantify precipitated protein
111. Remove samples from the incubator, and centrifuge 13300 rpm, 5 min 
SECTION: BCA assay to quantify precipitated protein
114. Calculation refers to the section: Calculate protein content per filter
SECTION: BCA assay to quantify precipitated protein
112. Each microplate can hold eight standard solutions and forty samples+blanks, all in duplicate
 

SECTION: BCA assay to quantify precipitated protein
113. 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: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
80. Cool down samples to room temperature, and centrifuge 13300 rpm, 5 min 
SECTION: Enhanced Pierce BCA assay for protein 5 to 200 ug/sample
83. Calculation refers to the section: Calculate protein content per filter
SECTION: Protein extraction
9.1. Rinse forceps with 95% ethanol and air dry
 

SECTION: Protein extraction
9.2. 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.
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9.3. 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

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9.4. Insert the strip into beads mixture.
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9.5. Place the bead tubes  on ice . 

SECTION: Reagent
4.5. Shake at    for complete dissolution.
 


SECTION: Crude protein quantitation
41. Thaw protein extract (  ) 
SECTION: BCA assay to quantify precipitated protein
100. Reverse pipetting, add 100 µL  RSB (  ) to each tube with precipitated protein (  ). 
Vortex for complete re-solubilization.
SECTION: BCA assay to quantify precipitated protein
99. Re-solubilization buffer (RSB):
Add 500 µL20% sarcosine (  ) into 9.5 mL 50 mM Tris ( ) 
SECTION: Protein precipitation
98. Store at -80 °C .

SECTION: BCA assay to quantify precipitated protein
105. Prepare seven 2 mL microtubes for PS2 to PC8.
SECTION: BCA assay to quantify precipitated protein
102. Organize eight 2 mL microtubes in the tube rack, label the tubes from PS1 to PS8, P denotes precipitated.
SECTION: BCA assay to quantify precipitated protein
104. Reverse pipet 100 µL  RSB (  ) into PS1.
SECTION: Calculate protein content per filter
62. Standard curve
SECTION: Calculate protein content per filter
62.1. For crude protein, the standard curve is quadratic.
  
Where   is the blank corrected absorbance at 562 nm and   is the concentration of protein.
SECTION: Calculate protein content per filter
62.2. For precipitated protein, the standard curve is linear. 
  
Where   is the blank corrected absorbance at 562 nm and   is the concentration of protein.
SECTION: Calculate protein content per filter
64.1. Enter the standard deviation (SD) of the absorbance values from the blank as   in the standard curve to determine the  .
SECTION: Calculate protein content per filter
64.2.   
SECTION: Calculate protein content per filter
66. If the calculated sample concentration is lower than 5 ug, then use "<LOD" as the result, indicating the protein content is undetectable.