TITLE: Chloroform-methanol protein precipitation from microalgae and Pierce BCA assay
AUTHORS: Ying-Yu Hu, Christopher Lord, Zoe V Finkel

[DESCRIPTION]
Chlorophyll, phospholipids, sucrose, glycerol and some detergent in crude protein extracted from microalgae can interfere the Pierce BCA protein assay. In order to remove these interference, bead miller extracted protein is precipitated by chloroform-methanol prior to BCA assay. The resulting precipitation is dissolved into Sarcosine-Tris solution. Low limit of detection is about 5 ug/mL.

[STEPS]
SECTION: Reagent preparation
1. Tris buffer  5 mM (pH 8.0)
Add 500 µL  1 MpH 8.0  Tris into 100 mL MilliQ water  
SECTION: Reagent preparation
2. 20% Sarcosine
Dilute 2 part 30%  N-lauroylsarcosine sodium salt with 1 part 5 mM (pH 8.0) Tris buffer
  
SECTION: Protein precipitation
3. Thaw protein extract 
SECTION: Protein precipitation
4. Turn on refrigerate centrifuge
 

SECTION: Protein precipitation
5. Turn on incubator/shaker, preheat to 37 °C 
 

SECTION: Protein precipitation
6. Prepare ice-bath
SECTION: Protein precipitation
7. Well mix the extract and then transfer 100 µL of extract to 2 mL microtube (Abdos tubes give better precipitation results), in replicate.

 

 

SECTION: Protein precipitation
8. In the fume hood, add 400 µL methanol
  
SECTION: Protein precipitation
9. Gently vortex for 30 s by using a tube insert
 

SECTION: Protein precipitation
10. In the fume hood, add 100 µL chloroform  
SECTION: Protein precipitation
11. Gently vortex for 30 s by using a tube insert
SECTION: Protein precipitation
12. In the fume hood, add 300 µL MilliQ
SECTION: Protein precipitation
13. Gently vortex for 30 s by using a tube insert
SECTION: Protein precipitation
14. Incubate  on ice for 30 min 

SECTION: Protein precipitation
15. 20000 rcf, 10 min, 4 °C 
SECTION: Protein precipitation
16. In the fume hood, remove upper phase by leaving about 250 µL liquid
 

SECTION: Protein precipitation
17. In the fume hood, add 300 µL methanol
SECTION: Protein precipitation
18. Gently mix the liquid until bottom layer disappear and the solution is homogenous. 
 

SECTION: Protein precipitation
19. 20000 rcf, 10 min, 4 °C 
SECTION: Protein precipitation
20. In the fume hood, remove all solvent.
 

SECTION: Protein precipitation
23. Dry pellet in vacuum desiccator for at least 30 min at Room temperature 
 

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

SECTION: BCA assay
26. BSA standard solutions
  
 

SECTION: BCA assay
28. 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
29. 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
27. Vortex and then use reverse pipetting: transfer 100 µL standard solutions into the corresponding tubes, except for SD1 (it has already been 100 uL).
SECTION: BCA assay
30. 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
31. Vortex each tube, shake and incubate at 37 °C for 30 min 
SECTION: BCA assay
32. Remove samples from the incubator.
SECTION: BCA assay
33. Load samples into microplate in duplicate:
 

 

SECTION: BCA assay
34. 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: Calculation
35. Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard.
SECTION: Calculation
36. Subtract the average 562 nm absorbance measurement of the blank sample (filter) replicates from the 562 nm measurements of all other individual sample.
SECTION: Calculation
37. 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.
  
SECTION: Calculation
38. 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.
SECTION: Calculation
39. 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.
SECTION: BCA assay
25. Use tube insert, vortex all tubes for 15 to 30 min until pellet is completely re-dissolved.
SECTION: Protein precipitation
21. If pellet tends to be aspired with solvent, add another 300 µL  methanol, gently vortex, and 20000 rcf, 10 min, 4 °C 

SECTION: Protein precipitation
22. 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.