Assessing enrichment of proteins in the mitochondrial fraction in HEK cells
OLIVIA HARDING
Abstract
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.
Before start
Add protease and phosphatase inhibitors such as Pepstatin A, TAME, Leupeptin, DTT, and PMSF) to Reagents A and C (800 uL Reagent A and 1300 uL Reagent C needed per sample)
Chill all reagents and tools on ice before starting.
Steps
Cell Lysis and Mitochondrial Enrichment
Place dish of cells on ice and gently aspirate media.
Add 800 uL cold PBS and scrape cells into a 2 mL tube
If using multiple plates per condition, combine all cells into 800 uL PBS.
Save 50 uL of cell suspension for whole cell lysis (Input).
Centrifuge both tubes at 850g for 2 min at 4 degrees C.
Aspirate supernatants from both tubes.
For Input sample, snap freeze in liquid nitrogen and store at -20 degrees C for later lysis.
For bulk of cells, add 800 uL Reagent A with protease and phosphatase inhibitors.
Vortex 5 sec and incubate on ice for 2 min
Add 10 uL Reagent B
Incubate on ice for 5 min, vortexing every minute.
Add 800 uL Reagent C with protease and phosphatase inhibitors
Invert by hand to mix
Centrifuge at 700 g for 10 min at 4 degrees C.
Transfer supernatant to a new tube
Centrifuge at 3,000 g for 15 min at 4 degrees C
Transfer supernatant (cytosolic fraction) to a new tube and save on ice.
Add 500 uL Reagent C with protease and phosphatase inhibitors to the pellet
Centrifuge 12,000 g for 5 min at 4 degrees C
Save supernatant as Wash.
Suspend final pellet in 80 uL RIPA buffer with protease and phosphatase inhibitors by vigorous pipetting and vortexing.
Save sample on ice or at -20 degrees C. Minimize freeze/thaw cycles.
Prepare Input lysis
Thaw snap-frozen whole cell sample on ice for 5 min
Add 100 uL RIPA buffer with protease and phosphatase inhibitors
Incubate on ice 20 min
Centrifuge 20 min at 17,000 g at -20 degrees C
Save supernatant as Input
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
Gel Electrophoresis and transfer
Add denaturing buffer to samples
Load 30 ug of each sample onto acrylamide gels along with a molecular weight standard for each set
30 ug into 10% gel and 30 ug into 14% gel for each sample
Run samples and transfer resulting protein array to PVDF membrane
Membrane blotting
Stain total protein and image with preferred method
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)
Clear total protein stain
Block membranes
Incubate membranes with primary antibodies for the respective proteins overnight at 4 degrees.
90 kDa - 260 kDa : LAMP1, TBK1
40 kDa - 70 kDa : Parkin
25 kDa - 38 kDa : GAPDH
(14% set)
8 kDa - 25 kDa : TOMM20
Wash membranes 4x 5 min with TBS and 0.2% TWEEN
Incubate membranes with LICOR infrared spectrum secondary antibodies to the respective species
Wash membranes 4x 5 min with TBS and 0.2% TWEEN
Image membranes with Odyssey scanner
Quantification
Use ImageStudio sofware to outline bands of interest for each sample with the Box function
Assess GAPDH bands in Enrichment fraction to determine amount of cytosolic contamination. Assess LAMP1 bands to determine amount of lysosomal contamination.
Transfer band intensities after background subtraction to an Excel document
Divide band intenities from the Enrichment fraction by their corresponding intensities in the Input section to determine the enrichment efficacy.
