Jul 31, 2024

Public workspaceGeneral Total Protein Sample Preparation Protocol for the Immunodetection of Auxenochlorella protothecoides Proteins.

DOI
dx.doi.org/10.17504/protocols.io.dm6gpzdm8lzp/v1
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
  • Merchant Lab UC Berkeley
Dimitrios J. Camacho
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpzdm8lzp/v1
Protocol Citation: Dimitrios J. Camacho, Sabeeha S. Merchant 2024. General Total Protein Sample Preparation Protocol for the Immunodetection of Auxenochlorella protothecoides Proteins.. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzdm8lzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 25, 2024
Last Modified: July 31, 2024
Protocol Integer ID: 102337
Keywords: Protein, total protein, imumunodetection, Auxenochlorella
Funders Acknowledgements:
National Institutes of Health (NIH): Nutritional Copper Signaling and Homeostasis Grant
Grant ID: GM 042143
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
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.
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.
Materials
  1.      Metal free 15 mL tubes - Globe Scientific Inc. Centrifuge, high performance, red screw cap, assembled, polypropylene. Cat. No. 6295, with a maximum rating of 17,000 ×g.
  2.      Metal free 50 mL tubes - Globe Scientific Inc. Centrifuge, high performance, red screw cap, assembled, polypropylene. Cat. No. 6297, with a maximum rating of 20,000 ×g.
  3.      1 L HDPE bottle, 4 bottles.
  4.      Ultra-pure 6 M HCl.
  5.      Ultra-pure ICP-MS grade Milli-Q H2O.
  6.      Trace metal grade Na2HPO4 anhydrous (dibasic, MW = 138 g/mol).
  7.      Trace metal grade NaH2PO4• H2O (monobasic, MW = 138 g/ mol).
  8.      cOmplete‱ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail.
  9.      1-10 L dewar of liquid nitrogen.
  10.   1.5 mL metal free screw cap tubes with gasket. 
  11.   1.5 mL metal free conical tubes. 
  12.   Acid washed, metal free glass beads 425-600 µm.
  13.   Acid washed, metal free glass beads 4 mm. 
  14.   Centrifuge.
  15.   Sterile hood.
  16.   Biospec Mini-BeadBeater-16
  17.   Liquid nitrogen flash freezing tube rack.
  18.   RAININ P100, P1000 pipettes and tips. 
Safety warnings
Liquid nitrogen can displace oxygen and cause asphyxiation. If transporting greater than Amount10 L of liquid nitrogen, do not accompany the dewar in the elevator. Have someone take the dewar out of the elevator at your floor. Do not cap tubes tightly after flash freezing in liquid nitrogen. Tubes may explode if capped when they warm up. Do not use nitrile or rubber gloves to handle liquid nitrogen. Use cryogenic gloves, closed toe shoes, a lab coat, a cryogenic apron, and a face shield when handling liquid nitrogen.

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 waterTemperature0 °C .
 
 2. Fill a 1-10 L dewar of liquid nitrogen. 
 
 3. Prepare cell lysis tubes.
3.1. Add Amount200 mg of 425-600 µm acid washed glass beads to a Amount1.5 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 Concentration10 millimolar (mM) sodium-phosphate solution, Ph7 and protease inhibitor cocktail mixture.
4.1. Acid wash the Amount1 L HDPE bottles (Quinn and Merchant, 1998),(Camacho and Merchant, 2024). 
4.2. Make Concentration1 Molarity (M) NaH2PO4 by adding Amount138 g of NaH2PO4• H2O to a Amount1 L bottle with stirring and fill to Amount1 L with Milli-Q H2O. Store at 4 °C. 
4.3. Make Concentration1 Molarity (M) Na2HPO4 by adding Amount142 g of Na2HPO4 (anhydrous) to a Amount1 L bottle with stirring and fill toAmount1 L with Milli-Q H2O. Store at 4 °C.
4.4. Make Concentration1 Molarity (M) sodium-hosphate solution, Ph7 by mixing Amount390 mL of Concentration1 Molarity (M) NaH2PO4 and Amount610 mL of Concentration1 Molarity (M) Na2HPO4. Store at 4 °C.
4.5. Dilute Concentration1 Molarity (M) sodium-phosphate, Ph7 toConcentration10 millimolar (mM) sodium-phosphate, Ph7 by adding Amount10 mL of Concentration1 Molarity (M) sodium-phosphate, Ph7 to an acid washed HDPE bottle containing Amount990 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 Amount15 mL tube.  
4.7. Add 1 cOmplete‱ ULTRA protease inhibitor cocktail tablet to Amount10 mL of Concentration10 millimolar (mM) sodium-phosphate, Ph7 . Keep the cocktail on wet ice. 

Collect 108 - 109 cells by centrifugation (Centrifigation10000 x g, 4°C, 00:02:00 ) using Globe Scientific metal free Amount15 mL or Amount50 mL tubes. Discard the supernatant.

2m
Wash the cells by resuspending the cell pellet in Amount400 µL of trace metal grade Concentration10 millimolar (mM) sodium-phosphate, Ph7 and protease mixture. Collect cells by centrifugation (see step 1) and remove the supernatant with a P1000 pipette tip.

Resuspend the cells in Amount300 µL of trace metal grade Concentration10 millimolar (mM) sodium-phosphate, Ph7 and protease mixture. Transfer the cell suspension to cold Amount1.5 mL 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 Amount100 µL of the sodium-phosphate and protease mixture to wash the sides of the tube and transfer all material to the respective Amount1.5 mL tube containing glass beads. Cells may stick to the P1000 tip so use a P100 to add the extra Amount100 µL of sodium-phosphate and protease mixture.

Optional: Flash freeze cell suspension in liquid nitrogen and store in Temperature-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 Duration00:00:10 .

10s
Store samples in Temperature-80 °C until further processing.

Protocol references
Camacho, D. J., Perrino, C., and Merchant, S. S. (2024) HEPES-phosphate medium for growth of Auxenochlorella protothecoides, suitable for studies of trace element nutrition. Protocols.io 
 
Quinn, J. M., & Merchant, S. (1998). [18] Copper-responsive gene expression during adaptation to copper deficiency. In Methods in enzymology (Vol. 297, pp. 263-279). Academic Press.