Chloroform-methanol protein precipitation from microalgae and Pierce BCA assay

Tris buffer 5 (pH 8.0)

Add 500µL 1``8.0 Tris into 100 mL MilliQ water

20% Sarcosine

Dilute 2 part 30% N-lauroylsarcosine sodium salt with 1 part 5 (pH 8.0) Tris buffer

Thaw protein extract

Turn on refrigerate centrifuge

Equipment

Value	Label
CENTRIFUGE 5430 R	NAME
Eppendorf	BRAND
MP2231000510	SKU

Turn on incubator/shaker, preheat to 37°C

Equipment

Value	Label
SHAKING INCUBATOR	NAME
71L	TYPE
Corning® LSE™	BRAND
6753	SKU

Prepare ice-bath

Well mix the extract and then transfer 100µL of extract to 2 mL microtube (Abdos tubes give better precipitation results), in replicate.

Equipment

Value	Label
Micro Centrifuge Tubes	NAME
Abdos	BRAND
P10203	SKU

In the fume hood, add 400µL methanol

Gently vortex for 0h 0m 30s by using a tube insert

Equipment

Value	Label
VWR ANALOG VORTEX MIXER	NAME
VWR	BRAND
10153-838	SKU
With tube insert	SPECIFICATIONS

In the fume hood, add 100µL chloroform

Gently vortex for 0h 0m 30s by using a tube insert

In the fume hood, add 300µL MilliQ

Gently vortex for 0h 0m 30s by using a tube insert

Incubate On ice for 0h 30m 0s

20000rcf,4°C

In the fume hood, remove upper phase by leaving about 250µL liquid

In the fume hood, add 300µL methanol

Gently mix the liquid until bottom layer disappear and the solution is homogenous.

20000rcf,4°C

In the fume hood, remove all solvent.

If pellet tends to be aspired with solvent, add another 300µL methanol, gently vortex, and 20000rcf,4°C

In the fume hood, remove most solvent by using 1000 uL pipet tip, and then remove the rest by using 100 uL pipet tip. Do not remove pellet with solvent.

Dry pellet in vacuum desiccator for at least 0h 30m 0s at Room temperature

Add 5µL 20% sarcosine and 95µL 5 (pH 8.0) Tris buffer to dry protein pellet, incubate at 37°C for 15 to 30 min.

Use tube insert, vortex all tubes for 15 to 30 min until pellet is completely re-dissolved.

BSA standard solutions

A	B	C	D	E
SD1	5	95	0	0
SD2	25	470	5	0.02
SD3	25	463	12	0.048
SD4	25	450	25	0.1
SD5	25	425	50	0.2
SD6	25	375	100	0.4
SD7	25	275	200	0.8
SD8	25	225	250	1

Vortex and then use reverse pipetting: transfer 100µL standard solutions into the corresponding tubes, except for SD1 (it has already been 100 uL).

Use the following formula to determine the total volume of working reagent (WR) required. Consider leaving several mL of extra volume:

(# standards + # samples) 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

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

Remove samples from the incubator.

Load samples into microplate in duplicate:

Equipment

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

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. The standard curve is quadratic.

For the calculation convenience, plot BSA concentration (Conc) versus Corrected absorbance (Abs) to obtain a standard curve as following:

Conc_mg/mL = a X Abs^2 + b X Abs + c

Use the corrected measured absorbance of samples (Abs) to calculate the total protein concentration (Conc_mg/mL) from each sample.

Protein_mg/filter = Conc_mg/mL X PEB_mL

Where PEB is the volume of protein extraction buffer used to extract protein from microalgae sample.