General Total Protein Sample Preparation Protocol for the Immunodetection of Auxenochlorella protothecoides Proteins.
Sabeeha S. Merchant, Dimitrios Camacho
Abstract
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.
- Prepare a bucket of wet ice. Add water to the ice so that the tubes will be in contact with the ice water
0°C.
2. Fill a 1-10 L dewar of liquid nitrogen.
3. Prepare cell lysis tubes.
3.1. Add `200mg` of 425-600 µm acid washed glass beads to a `1.5mL` screw cap tube with gaskets.
3.2. Add one 4 mm glass bead to the tube. Keep the tubes on wet ice.
-
Make the trace metal grade
10millimolar (mM)sodium-phosphate solution,7and protease inhibitor cocktail mixture.4.1. Acid wash the
1LHDPE bottles (Quinn and Merchant, 1998),(Camacho and Merchant, 2024).4.2. Make
1Molarity (M)NaH2PO4 by adding138gof NaH2PO4• H2O to a1Lbottle with stirring and fill to1Lwith Milli-Q H2O. Store at 4 °C.4.3. Make
1Molarity (M)Na2HPO4 by adding142gof Na2HPO4 (anhydrous) to a1Lbottle with stirring and fill to1Lwith Milli-Q H2O. Store at 4 °C.4.4. Make
1Molarity (M)sodium-hosphate solution,7by mixing390mLof1Molarity (M)NaH2PO4 and610mLof1Molarity (M)Na2HPO4. Store at 4 °C.4.5. Dilute
1Molarity (M)sodium-phosphate,7to10millimolar (mM)sodium-phosphate,7by adding10mLof1Molarity (M)sodium-phosphate,7to an acid washed HDPE bottle containing990mLMilli-Q H2O. Store at 4 °C.4.6. Right before sampling, make a fresh sodium-phosphate protease inhibitor cocktail mixture in a metal free
15mLtube.4.7. Add 1 cOmpleteTM ULTRA protease inhibitor cocktail tablet to
10mLof10millimolar (mM)sodium-phosphate,7. Keep the cocktail on wet ice.
Steps
Collect 108- 109 cells by centrifugation (10000x g,4°C) using Globe Scientific metal free 15mL or 50mL tubes. Discard the supernatant.
Wash the cells by resuspending the cell pellet in 400µL of trace metal grade 10millimolar (mM) sodium-phosphate, 7 and protease mixture. Collect cells by centrifugation (see step 1) and remove the supernatant with a P1000 pipette tip.
Resuspend the cells in 300µL of trace metal grade 10millimolar (mM) sodium-phosphate, 7 and protease mixture. Transfer the cell suspension to cold 1.5mL screw cap tubes containing acid washed beads.
The suspension will be thick and sticky. To collect the rest of the cell suspension, add an additional 100µL of the sodium-phosphate and protease mixture to wash the sides of the tube and transfer all material to the respective 1.5mL tube containing glass beads. Cells may stick to the P1000 tip so use a P100 to add the extra 100µL of sodium-phosphate and protease mixture.
Optional: Flash freeze cell suspension in liquid nitrogen and store in -80°C
Carefully fill a tube rack with liquid nitrogen to a level where half of the tube is submerged.
Make sure tubes are not tightly closed and air is allowed to pass through with the cap on.
Use tongs to place samples into the liquid nitrogen for 0h 0m 10s.
Store samples in -80°C until further processing.
