TITLE: Total protein in microalgae: Pierce BCA protein assay
AUTHORS: Yingyu Hu, Zoe V. Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe a protocol for extracting total protein from microalgae and quantifying total protein by Pierce BCA protein assay.</div><br/><div class = "text-block"><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf</span></a></div></div>

[STEPS]
?. [Sample collection]
Calculate the volume to obtain enough biomass for the assay:Minimum sampling volume (mL) = 750/(Chl-a_ug/L), if protein is extracted by 500 ul extraction buffer.
?. [Sample collection]
Use 5 inches Hg to filter microalgae samples onto polycarbonate filters (pore size varies).
?. [Sample collection]
Fold filter in quarter and place into an MP biomedical bead tube.
Use 2 ml lysing matrix tube for 25 mm filter; 15 ml Teenprep tube for 47 mm filterLarge ceramic beads are recommended for diatoms and larger phytoplankton cells (2 mL: MP116913500/ 15 mL: MP116933050)Small yttrium beads are recommended for picoplankton (2 mL: MP116960100/ 15 mL: MP116975050)
?. [Sample collection]
Flash-freeze tubes in thermo flask with liquid nitrogen
?. [Sample collection]
Store at  until further processing
-80 °C
?. [Prepare protein solubilization buffer (PSB)]
?. [Prepare protein solubilization buffer (PSB)]
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
?. [Prepare protein solubilization buffer (PSB)]
For each 10 g PSB
?. [Prepare protein solubilization buffer (PSB)]
Use antistatics weighing dish to weigh the following chemicals:(1) 0.136 g Tris base(2) 0.133 g Tris HCl(3) 0.8 g Lithium dodecyl sulphate
?. [Prepare protein solubilization buffer (PSB)]
Place a plastic beaker on the top of the scale surface
?. [Prepare protein solubilization buffer (PSB)]
Remove the cap of a 15 mL tube and sit it in the beaker
?. [Prepare protein solubilization buffer (PSB)]
Tare the total weight of beaker and tube
?. [Prepare protein solubilization buffer (PSB)]
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
?. [Prepare protein solubilization buffer (PSB)]
Use a transfer pipet to add 4 g glycerol into the tube
?. [Prepare protein solubilization buffer (PSB)]
Add 40 ul 0.5 M EDTA into the tube
?. [Prepare protein solubilization buffer (PSB)]
Top to 10 g with MilliQ water
?. [Prepare protein solubilization buffer (PSB)]
Mix by shaker until all solutes are completely dissolved.
Prepare this solution in advance, since it takes some time to have solutes dissolved, especially LDS.
?. [Prepare Pefabloc solution]
Pefabloc is a protease inhibitor, and it loses activity over 24 hours.
?. [Prepare Pefabloc solution]
Add 20.86 mL MilliQ into 100 mg Pefabloc to obtain a final concentration of 20 mM.
?. [Prepare Pefabloc solution]
Aliquot into 2.5 ml portions and keep frozen at
-20 °C
?. [Prepare Pefabloc solution]
The solution can be frozen~thawed multiple times.
?. [Assay Day 1: Extract protein from microalgae]
Prepare protein extraction buffer (PEB):Each 1 mL PEB has250 ul PSB20 ul 20 mM Pefabloc730 ul MilliQ water
?. [Assay Day 1: Extract protein from microalgae]
Prepare ice-bath, keep all samples in the ice-bath
?. [Assay Day 1: Extract protein from microalgae]
If samples and blank filters are already in bead tubes, add 1 mL PEB onto the filter directly.
Volume of PEB varies due to the actual biomass collected (see guideline)
?. [Assay Day 1: Extract protein from microalgae]
If samples are in cryo tubes instead of bead tubes
?. [Assay Day 1: Extract protein from microalgae]
Rinse forceps with 70% ethanol and air dry
?. [Assay Day 1: Extract protein from microalgae]
Add 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.
Volume of PEB varies due to the actual biomass collected (see guideline)
?. [Assay Day 1: Extract protein from microalgae]
Label bead tubes and use clean forceps to transfer samples and blank filters into its corresponding bead tube.
?. [Assay Day 1: Extract protein from microalgae]
Turn on FastPrep
?. [Assay Day 1: Extract protein from microalgae]
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.
?. [Assay Day 1: Extract protein from microalgae]
Run 1 min at 6.5 m/s
?. [Assay Day 1: Extract protein from microalgae]
Keep tubes  for
on ice
?. [Assay Day 1: Extract protein from microalgae]
Check labels. Put tubes back into FastPrep.
?. [Assay Day 1: Extract protein from microalgae]
Run 1 min at 6.5 m/s
?. [Assay Day 1: Extract protein from microalgae]
Keep tubes  for
on ice
?. [Assay Day 1: Extract protein from microalgae]
Check labels. Put tubes back into FastPrep.
?. [Assay Day 1: Extract protein from microalgae]
Run 1 min at 6.5 m/s
?. [Assay Day 1: Extract protein from microalgae]
Keep tubes   for
on ice
?. [Assay Day 1: Extract protein from microalgae]
Check labels. Put tubes back into FastPrep.
?. [Assay Day 1: Extract protein from microalgae]
Run 1 min at 6.5 m/s
?. [Assay Day 1: Extract protein from microalgae]
Turn on refrigerated centrifuge
?. [Assay Day 1: Extract protein from microalgae]
Keep tubes  for
on ice
?. [Assay Day 1: Extract protein from microalgae]
Centrifuge:2 mL Lysing Matrix tubes at 15 mL Teenprep tube at
Centrifuge: 14800 34, 5 min, 4 10
Centrifuge: 6500 34, 5 min, 4 10
?. [Assay Day 1: Extract protein from microalgae]
Transfer all supernatant to a 1.7 mL microtube
For the extraction with 500 ul PEB in Teenprep tube, use 200 ul tip and go straight to the bottom from the side.
?. [Assay Day 1: Extract protein from microalgae]
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 mM pefabloc and transfer 150 ul to a 600 ul mcirotube. Put the rest of the stock back into the freezer immediately.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Organize eight 1.7 mL microtubes in the tube rack, label the tubes from SD1 to SD8.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Use 0.5 mL Finntip Stepper Tip to dispense 10 ul pefabloc into each microtube
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Use 1.25 mL Finntip Stepper Tip (Thermo Fisher 9404180) to dispense 125 ul PSB into each microtube.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Add MilliQ into each microtube according to the sheet below:  ABCDEF1   Standard  Pefabloc (20 mM) (µL)PSB (µL)  MQ (uL)    BSA (2mg/mL) (uL)    Final Conc. (mg/mL)  2  SD1    10    125    365    0    0  3  SD2    10    125    360    5    0.02  4  SD3    10    125    353    12    0.048  5  SD4    10    125    340    25    0.1  6  SD5    10    125    315    50    0.2  7  SD6    10    125    265    100    0.4  8  SD7    10    125    165    200    0.8  9  SD8    10    125    115    250    1
ABCDEF1   Standard  Pefabloc (20 mM) (µL)PSB (µL)  MQ (uL)    BSA (2mg/mL) (uL)    Final Conc. (mg/mL)  2  SD1    10    125    365    0    0  3  SD2    10    125    360    5    0.02  4  SD3    10    125    353    12    0.048  5  SD4    10    125    340    25    0.1  6  SD5    10    125    315    50    0.2  7  SD6    10    125    265    100    0.4  8  SD7    10    125    165    200    0.8  9  SD8    10    125    115    250    1
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Break the ampule of BSA standard
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Reverse pipet certain amount of BSA (2 mg/mL) into each tube according to the sheet
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Vortex each tube.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Dispense 4 uL of each standard solution onto microdrop plate.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Read absorbance of eight standard solutions at 205 nm
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Subtract absorbance at 205 nm of blank standard from the 205 nm measurements of all other standard solutions
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Plot the blank-corrected 205 nm measurement for each standard solution versus its concentration in mg/ml.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
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 passes the screen.
?. [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 since Finntip stepper is unable to expel the entire volume from the tip:(# standards + # samples + # blank filters) X (# replicates) X (200 ul) = total volume WR required
?. [Assay Day 2: Prepare BCA working reagent (WR)]
Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA Reagent B in a 50 mL falcon tube
?. [Assay Day 2: Pierce BCA assay]
Turn on incubator and preheat to
37 °C
?. [Assay Day 2: Pierce BCA assay]
Prepare 96-well microplate and lid: Each microplate can load eight standard solutions and forty samples+blanks, duplicated
Pipet tip is for single use only.
?. [Assay Day 2: Pierce BCA assay]
?. [Assay Day 2: Pierce BCA assay]
Reverse pipette 25 ul of each standard or sample/blank replicate into the microplate well.
?. [Assay Day 2: Pierce BCA assay]
Use 2.5 mL Finntip stepper tip to load 200 ul WR, starting from blank, including blank of standard solution and blank of the samples.
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
Dispense WR column by column.Refill the stepper in between two columns of wells.
?. [Assay Day 2: Pierce BCA assay]
Cover plate with lid.
?. [Assay Day 2: Pierce BCA assay]
Shake and incubate at  for
37 °C
?. [Assay Day 2: Pierce BCA assay]
Read absorbance at 562 nm
?. [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.
?. [Calculate protein content per filter]
Subtract the average 562 nm absorbance measurement of the blank sample (filter) replicates from the 562 nm measurements of all other individual sample.
?. [Calculate protein content per filter]
Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard versus its concentration in mg/ml.
?. [Calculate protein content per filter]
Use the standard curve to determine the protein concentration of each unknown sample by using its blank-corrected 562 absorbance.
?. [Calculate protein content per filter]
?. [Calculate protein content per filter]
Protein_mg/filter = Protein_mg/mL X PEB_mL
?. [Assay Day 1: Extract protein from microalgae]
Centrifuge at   to completely remove debris.
Centrifuge: 14800 34, 5 min, 4 10
?. [Assay Day 1: Extract protein from microalgae]
Transfer supernatant to microtube, avoid any debris.
?. [Assay Day 2: Pierce BCA assay]
Transfer extracted samples from  to
-80 °C
on ice
?. [Sample collection]
Freeze dry samples before processed.
?. [Assay Day 2: Prepare Bovine serum albumin (BSA) standard solutions]
Standard solutions can be kept at  .
0 Room temperature