TITLE: General Total Protein Sample Preparation Protocol for the Immunodetection of Auxenochlorella protothecoides Proteins.
AUTHORS: Dimitrios Camacho, Sabeeha S. Merchant

[DESCRIPTION]
This protocol describes a general method for quickly preparing and storing protein samples for the immunodetection of Auxenochlorella protothecoides proteins. The protocol was developed for metal free applications where the metal contents of Auxenochlorella protothecoides cells are of importance to the proteins studied. This protocol should be adapted to optimize sampling conditions for each protein of interest.

[BEFORE_START]
 1. Wipe down all work surfaces with 70% EtOH. 

2. Prepare a bucket of wet ice. Add water to the ice so that the tubes will be in contact with the ice water0 °C . 
 
 2. Fill a 1-10 L dewar of liquid nitrogen. 
 
 3. Prepare cell lysis tubes.
     3.1. Add 200 mg of 425-600 µm acid washed glass beads to a 1.5 mL screw cap tube with gaskets. 
     3.2. Add one 4 mm glass bead to the tube. Keep the tubes on wet ice.
 
4. Make the trace metal grade 10 millimolar (mM) sodium-phosphate solution, pH 7 and protease inhibitor cocktail mixture.
     4.1. Acid wash the 1 L HDPE bottles (Quinn and Merchant, 1998),(Camacho and Merchant, 2024). 
     4.2. Make 1 Molarity (M) NaH2PO4 by adding 138 g of NaH2PO4• H2O to a 1 L bottle with stirring and fill to 1 L with Milli-Q H2O. Store at 4 °C. 
     4.3. Make 1 Molarity (M) Na2HPO4 by adding 142 g of Na2HPO4 (anhydrous) to a 1 L bottle with stirring and fill to1 L with Milli-Q H2O. Store at 4 °C.
     4.4. Make 1 Molarity (M) sodium-hosphate solution, pH 7 by mixing 390 mL of 1 Molarity (M) NaH2PO4 and 610 mL of 1 Molarity (M) Na2HPO4. Store at 4 °C.
     4.5. Dilute 1 Molarity (M) sodium-phosphate, pH 7 to10 millimolar (mM) sodium-phosphate, pH 7 by adding 10 mL of 1 Molarity (M) sodium-phosphate, pH 7 to an acid washed HDPE bottle containing 990 mL Milli-Q H2O. Store at 4 °C.
     4.6. Right before sampling, make a fresh sodium-phosphate protease inhibitor cocktail mixture in a metal free 15 mL tube.  
     4.7. Add 1 cOmplete‱ ULTRA protease inhibitor cocktail tablet to 10 mL of 10 millimolar (mM) sodium-phosphate, pH 7. Keep the cocktail on wet ice. 


[GUIDELINES]
This protocol should be tailored to your specific protein of interest. For example, if you are also interested in light responsive proteins, you may wish to extract proteins in a specific light regime. 

Use only ICPMS grade trace metal free Ultra-pure ICP-MS grade Milli-Q H2O.
 
Review the certificates of analysis for each chemical used to verify potential metal contamination concentrations are minimized.

[STEPS]
1. Collect 108 - 109 cells by centrifugation (10000 x g, 2 min, 4 °C) using Globe Scientific metal free 15 mL or 50 mL tubes. Discard the supernatant.

4. Optional: Flash freeze cell suspension in liquid nitrogen and store in -80 °C

3. Resuspend the cells in 300 µL of trace metal grade 10 millimolar (mM) sodium-phosphate, pH 7 and protease mixture. Transfer the cell suspension to cold 1.5 mL screw cap tubes containing acid washed beads.

2. Wash the cells by resuspending the cell pellet in 400 µL of trace metal grade 10 millimolar (mM) sodium-phosphate, pH 7 and protease mixture. Collect cells by centrifugation (see step 1) and remove the supernatant with a P1000 pipette tip.

3.1. The suspension will be thick and sticky. To collect the rest of the cell suspension, add an additional 100 µL of the sodium-phosphate and protease mixture to wash the sides of the tube and transfer all material to the respective 1.5 mL tube containing glass beads. Cells may stick to the P1000 tip so use a P100 to add the extra 100 µL of sodium-phosphate and protease mixture. 

4.1. Carefully fill a tube rack with liquid nitrogen to a level where half of the tube is submerged.
4.2. Make sure tubes are not tightly closed and air is allowed to pass through with the cap on.
4.3. Use tongs to place samples into the liquid nitrogen for 10 s.

4.4. Store samples in -80 °C until further processing.