TITLE: Assessing enrichment of proteins in the mitochondrial fraction in HEK cells 
AUTHORS: OLIVIA  HARDING

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a method for measuring protein enrichment on mitochondria in various conditions. In the resulting Western blot, one can assess the level of contamination of other organelles in the enrichment prep. </div></div>

[STEPS]
?. [Cell Lysis and Mitochondrial Enrichment]
Place dish of cells on ice and gently aspirate media.
?. [Cell Lysis and Mitochondrial Enrichment]
Add 800 uL cold PBS and scrape cells into a 2 mL tube
?. [Cell Lysis and Mitochondrial Enrichment]
If using multiple plates per condition, combine all cells into 800 uL PBS.
?. [Cell Lysis and Mitochondrial Enrichment]
Save 50 uL of cell suspension for whole cell lysis (Input).
?. [Cell Lysis and Mitochondrial Enrichment]
Centrifuge both tubes at 850g for 2 min at 4 degrees C.
?. [Cell Lysis and Mitochondrial Enrichment]
Aspirate supernatants from both tubes.
?. [Cell Lysis and Mitochondrial Enrichment]
For Input sample, snap freeze in liquid nitrogen and store at -20 degrees C for later lysis.
?. [Cell Lysis and Mitochondrial Enrichment]
For bulk of cells, add 800 uL Reagent A with protease and phosphatase inhibitors.
?. [Cell Lysis and Mitochondrial Enrichment]
Vortex 5 sec and incubate on ice for 2 min
?. [Cell Lysis and Mitochondrial Enrichment]
Add 10 uL Reagent B
?. [Cell Lysis and Mitochondrial Enrichment]
Incubate on ice for 5 min, vortexing every minute.
?. [Cell Lysis and Mitochondrial Enrichment]
Add 800 uL Reagent C with protease and phosphatase inhibitors
?. [Cell Lysis and Mitochondrial Enrichment]
Invert by hand to mix
?. [Cell Lysis and Mitochondrial Enrichment]
Centrifuge at 700 g for 10 min at 4 degrees C.
?. [Cell Lysis and Mitochondrial Enrichment]
Transfer supernatant to a new tube
?. [Cell Lysis and Mitochondrial Enrichment]
Centrifuge at 3,000 g for 15 min at 4 degrees C
?. [Cell Lysis and Mitochondrial Enrichment]
Transfer supernatant (cytosolic fraction) to a new tube and save on ice.
?. [Cell Lysis and Mitochondrial Enrichment]
Add 500 uL Reagent C with protease and phosphatase inhibitors to the pellet
?. [Cell Lysis and Mitochondrial Enrichment]
Centrifuge 12,000 g for 5 min at 4 degrees C
?. [Cell Lysis and Mitochondrial Enrichment]
Save supernatant as Wash.
?. [Cell Lysis and Mitochondrial Enrichment]
Suspend final pellet in 80 uL RIPA buffer with protease and phosphatase inhibitors by vigorous pipetting and vortexing.
?. [Cell Lysis and Mitochondrial Enrichment]
Save sample on ice or at -20 degrees C. Minimize freeze/thaw cycles.
?. [Determine protein concentration of enrichment, cytosol, and input samples]
Perform a BCA assay with Pierce kit or similar using 10 uL sample per well in duplicate
?. Add 100 uL RIPA buffer with protease and phosphatase inhibitors
?. [Prepare Input lysis]
Thaw snap-frozen whole cell sample on ice for 5 min
?. Incubate on ice 20 min
?. Centrifuge 20 min at 17,000 g at -20 degrees C
?. Save supernatant as Input
?. [Gel Electrophoresis and transfer]
Add denaturing buffer to samples
?. [Gel Electrophoresis and transfer]
Run samples and transfer resulting protein array to PVDF membrane
?. [Gel Electrophoresis and transfer]
Load 30 ug of each sample onto acrylamide gels along with a molecular weight standard for each set
?. [Gel Electrophoresis and transfer]
30 ug into 10% gel and 30 ug into 14% gel for each sample
?. [Membrane blotting]
Stain total protein and image with preferred method
?. [Membrane blotting]
Cut membranes to produce sections for the respective proteins: (10% set)90 kDa - 260 kDa (LAMP1, TBK1)40 kDa - 70 kDa (Parkin)25 kDa - 38 kDa (GAPDH)(14% set)8 kDa - 25 kDa (TOMM20)
?. [Membrane blotting]
Clear total protein stain
?. [Membrane blotting]
Block membranes
?. [Membrane blotting]
Incubate membranes with primary antibodies for the respective proteins overnight at 4 degrees.90 kDa - 260 kDa : LAMP1, TBK140 kDa - 70 kDa : Parkin25 kDa - 38 kDa : GAPDH(14% set)8 kDa - 25 kDa : TOMM20
?. [Membrane blotting]
Wash membranes 4x 5 min with TBS and 0.2% TWEEN
?. [Membrane blotting]
Incubate membranes with LICOR infrared spectrum secondary antibodies to the respective species
?. [Membrane blotting]
Wash membranes 4x 5 min with TBS and 0.2% TWEEN
?. [Membrane blotting]
Image membranes with Odyssey scanner
?. [Quantification]
Use ImageStudio sofware to outline bands of interest for each sample with the Box function
?. [Quantification]
Transfer band intensities after background subtraction to an Excel document
?. [Quantification]
Divide band intenities from the Enrichment fraction by their corresponding intensities in the Input section to determine the enrichment efficacy.
?. [Quantification]
Assess GAPDH bands in Enrichment fraction to determine amount of cytosolic contamination. Assess LAMP1 bands to determine amount of lysosomal contamination.