Monitoring protein movements to and from the mitochondrion
in apoptosis, a high throughput quantitative solution
Petr Hájek, Nolan Priedigkeit, Jesse Mull, Jing Xie, Michael F. Marusich, Roderick A. Capaldi and James G. Murray
MitoSciences, Eugene, Oregon, 97403, USA

Figure 2: Method optimization for separation of cytosolic and mitochondrial fractions.

Mitosciences have developed asimple, rapid cell fractionation
method to obtain cytosol-,
mitochondriaand
nucleicontaining fractions. The method
involves sequential and selective
extraction of cytosolic and then mitochondrial proteins by detergents from a nucleus containing
fraction. This cell fractionation procedure can be performed either on cells in suspension or in ahigh throughput microplate format on adherent cells.

Optimization of cell permeabilization to separate cytosolic and mitochondrial fractions.

Figure 4: Quantitative ELISA analysis of cytochrome c release following Staurosporine
treatment in HeLa cells.
Quantitative ELISA analysis of cytochrome c release from the mitochondria into thecytosol in HeLa cells induced to undergo apoptosis by Staurosporine treatment.

Cell fractionations in a 96 well plate format are used to monitor translocation of Bax from the cytosol
to the mitochondria and cytochrome c and Smac from the mitochondria into the cytosol in HeLa
cells induced to undergo apoptosis by Staurosporine treatments. Cell fractionation followed by acytochrome c sandwich ELISA assay, offers a complete quantitative high throughput approach tomeasure cytochrome c release from mitochondria in cells undergoing apoptosis.

Product table
Antibodies

Applications

Species

Anti-AIF antibody [7F7AB10]
Anti-Cleaved PARP antibody [4B5BD2]
Anti-Cytochrome C antibody [37BA11]
Anti-Smac / Diablo antibody [8H5AA3]
Anti-Smac / Diablo antibody [Y12]
Anti-Bcl2 antibody
Anti-Bax antibody
Anti-PARP antibody
Anti-Endo G antibody
Anti-Bak antibody [Y164]
Anti-Bid antibody [3C5]Anti-Bad antibody [Y208]
Anti-Bim antibody
Anti-MCL1 antibody [8C6D4B1]
Anti-BNIP3L antibody
Anti-HtrA2 / Omi antibody
Anti-PUMA antibody
Anti-p53 antibody [PAb 240]

Flow Cyt, ICC/IF, IP, In-Cell ELISA, WB
Flow Cyt, ICC/IF, In-Cell ELISA, WB
Flow Cyt, ICC/IF, In-Cell ELISA, WB
Flow Cyt, ICC
Flow Cyt, ICC/IF, IHC-P, IP, WB
ICC, IHC-Fr, IHC-P, IP, WB
IHC-Fr, IHC-P, IP, WB
IHC-Fr, IHC-P, IP, WB
ICC/IF, IHC-P, WB
Flow Cyt, ICC, IHC-P, WB
ELISA, Flow Cyt, ICC/IF, IHC-P, WB
Flow Cyt, ICC/IF, IHC-P, IP, WB
IHC-P, WB
ELISA, Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WB
ICC/IF, IHC-P, WB
ICC/IF, IHC-FoFr, WB
ICC/IF, IHC-P, WB
ELISA, Flow Cyt, ICC/IF, IHC (Methanol fixed),
IHC-Fr, IHC-P, IP, WB

Hu
Hu
Hu, Ms, Rat, Ce, Cow
Hu
Hu, Ms, Rat
Hu, Ms, Rat, Cow
Hu, Ms, Rat
Hu, Ms, Rat
Hu, Ms, Rat
Hu, Ms, Rat
Hu
Hu, Ms, Rat
Hu
Hu, MsHu, MsHu, MsHu, MsApplications

Species

ApoTrack™ Cytochrome c Apoptosis WB
Antibody Cocktail
ApoTrack™ Cytochrome c Apoptosis ICC Antibody Kit

WB
In-Cell ELISA

Hu
Hu, Ms, Rat

Kits

Tests

Species

Cytochrome c Protein Quantity Microplate Assay Kit
p53 Human ELISA Kit
Mitochondrial Aldehyde Dehydrogenase (ALDH2)
Activity Assay Kit

1 x 96 tests
1 x 96 tests

Hu, Ms, Rat, Cow
Huab110172
ab117995

1 x 96 tests

Huab115348

Western blot antibody panels andICC antibody kits

Intoduction
The movement of pro-apoptotic factors of the Bcl2-family from the cytosol to the mitochondria
and the consequent permeabilization of the mitochondrial outer membrane to releasecytochrome c and Smac from the mitochondrial intermembrane space into the cytosol or AIF
to the nucleus are now well characterized steps in programmed cell death. However, there areconcurrent movements of other proteins, including kinases and transcription factors (e.g. p53),
to and from the organelle to both signal and modulate apoptosis.
The identification and quantification of these protein movements between cellularcompartments is necessary to fully understand and to differentiate between different pathwaysof cell death. Biochemical cell fractionation approaches mostly utilize mechanical cell
disruptions which are often difficult to standardize, require a relatively large amount of cellsample and the lack of multiple instruments often limits these methods to process one sample
at a time making it time consuming and difficult to perform on a large number of samples. They
also carry the risk of disrupting the mitochondrial membranes leading, in particular, to artificial
release of mitochondrial intermembrane space pro-apoptotic proteins. The mechanical cell
disruption is often incomplete and thus a removal of unbroken cells is frequently required. This
leads to losses of uncharacterized cell material which is difficult to account for.

Cytosolic (C) and mitochondrial (M) fractions of HeLa cells, seeded at 30,000 per well of a 48well plate were prepared using the Cell Fractionation HT method and variable concentrationsof Buffer A . Fractions were analyzed by western blotting using MitoSciences ApoTrack™
Cytochrome c Apoptosis WB Antibody Cocktail (ab110415) and supplemented with
antibodies against an additional cytosolic (Bax) marker, followed by appropriate HRPconjugated goat secondary antibodies and ECL detection. The Blue box indicates the dilution
of Buffer A for the optimal separation of cytosolic and mitochondrial proteins

Figure 3: Movement of proteins following Staurosporine treatment in HeLa cells.
Cytochrome c and Smac are released from the mitochondria into the cytosol and Bax
re-localizes from the cytosol to mitochondria during apoptosis induced by
Staurosporine treatment in HeLa cells.

The Mitosciences cell fractionation kit (ab109718) is based on a selective and sequential
extraction of cytosolic and mitochondrial proteins thus eliminating the need of mechanical cell
disruption and differential centrifugation. This poster presents a new generation of thismethodology specifically designed to fractionate adherant cells directly, without theirdetachment, in a high throughput microplate format. We demonstrate the utility of this highthroughput fractionation on following the movement of Bax, cytochrome c and Smac in HeLa
cells induced to undergo apoptosis by Staurosporine treatment using western blotting. Whencoupled to a microplate sandwich ELISA assay, these methodologies also present a completehigh throughput solution, as shown on monitoring of the cytochrome c release in apoptosis.

Cytosolic (C), mitochondrial (M) and nuclear (N) fractions of HeLa cells treated for 4 hrs with0.00, 0.02, 0.06, 0.18, 0.54, 1.62, 4.86 and 14.58 µM Staurosporine (A and B) or with 0.0 and
1.0 mM Staurosporine (C, D, E) were prepared using the Cell Fractionation HT method(ab109718). Fractions, each derived from one well of a 96-well plate, were analyzed by
MitoSciences ELISA Cytochrome c Protein Quantity Microplate Assay Kit (ab110172)(panels A, C and D). Parallel analyses of fractions prepared independently and thus showing
the inter-assay variation of the Cell Fractionation HT method are shown in C and D. Western
blot analyses of cytochrome c using MitoSciences ApoTrack™ Cytochrome c Apoptosis
WB Antibody Cocktail (ab110415) are shown for comparison (B and E). Data represent mean+/- standard error of the mean, n=4 (A and C), n=3 (D), n=2 (E), n=1 (B).

Conclusions• The HT method is designed for parallel fractionation of a large number of small samples ofadherant cells in a microplate format. It allows a preparation of cytosolic, mitochondrial andnuclear fractions from 96 cell samples in one hour.
• The fractions prepared by this method are particularly suitable for high throughput sandwich
ELISA microplate assays and also for Western blotting.

Figure 1: Cell Fractionation Kits HT Method• Validation of the Cell Fractionation HT methodology on the proteins known to translocate
between cytosol and mitochondria in cells undergoing apoptosis:

Cell Fractionation HT method (ab109718): showing fractionation of adherant cells intocytosol-, mitochondria- and nuclei-containing fractions.

• Staurosporine concentration-dependent release of cytochrome c (EC50 = 0.40 mM) and Smac(EC50 = 0.52 mM) from the mitochondria into the cytosol.
• Staurosporine concentration-dependent translocation of Bax (EC50 = 0.37 mM) from thecytosol into the mitochondria.
• Cleavage of nuclear-localized full length PARP.

Cells, grown in a microplate, are treated with Buffer
A to permeabilize the plasma membrane and to releasecytosolic proteins into the extraction buffer. The cytosoldepleted cells are then treated with Buffer B to extract themitochondrial proteins. Finally, the cytosol and mitochondria-depletedcells are treated with Buffer C to extract nuclear proteins.

Discover more at abcam.com

HeLa cells were treated for 4 hrs with concentrations of 0.00, 0.02, 0.06, 0.18, 0.54, 1.62, 4.86
and 14.58 µM Staurosporine. Cytosolic (C), mitochondrial (M) and nuclear (N) fractions, were
each derived from one well of a 96-well plate, prepared using the Cell Fractionation HT method.
Mobilization of proteins was analyzed by western blotting using MitoSciences ApoTrack™
Cytochrome c Apoptosis WB Antibody Cocktail (ab110415) and supplemented with anantibody against Smac, as well as with antibodies against Bax and PARP. Representative
blots as well as the quantitative analysis (mean +/- standard error, n=2) are shown.

Datasheet abcam.com/..

Hu, Ms, Rat

Cell Fractionation Kits

ab26

Datasheet abcam.com/..

ab110415
ab110417

Datasheet abcam.com/..

Datasheet abcam.com/...

Cell Fractionation Kit Standard
Cell Fractionation Kit - HT
Mitochondria Isolation Kit for Tissue
Mitochondria Isolation Kit for Tissue (with Dounce Homogenizer)
Mitochondria Isolation Kit for Cultured Cells
Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer)

ab109719
ab109718
ab110168
ab110169
ab110170
ab110171

LysatesAmounts

Applications

SpeciesBrain mitochondrial lysate
Brain mitochondrial lysate
Brain mitochondrial lysate
Brain mitochondrial lysate
Heart mitochondrial lysate
Heart mitochondrial lysate
Heart mitochondrial lysate
Heart mitochondrial lysate
Heart mitochondrial lysate
Heart mitochondrial lysate
Liver mitochondrial lysate
Liver mitochondrial lysate
Liver mitochondrial lysate
Liver mitochondrial lysate
Liver mitochondrial lysate

50 µg
2 mg
50 µg
2 mg
2 mg
50 µg
50 µg
2 mg
50 µg
2 mg
50 µg
50 µg
2 mg
2 mg
50 µg

SDS-Page, WBImmunocapture, BNPageSDS-Page, WB
Immunocapture, BNPage
Immunocapture, BNPageSDS-Page, WB
SDS-Page, WB
Immunocapture, BNPageSDS-Page, WB
Immunocapture, BNPageSDS-Page, WB
SDS-Page, WB
Immunocapture, BNPage
Immunocapture, BNPageSDS-Page, WB

Ms
Ms
Rat
Rat
Cow
Hu
Ms
Ms
Rat
Rat
Hu
Ms
Ms
Rat
Rat

Datasheet abcam.com/...
ab110345
ab110351
ab110342
ab110348
ab110338
ab110337
ab110344
ab110350
ab110341
ab110347
ab110339
ab110343
ab110349
ab110346
ab110340

Featured products
Anti-MTCO1 antibody [1D6E1A8]- Mitochondrial Loading
Control (ab14705)
Applications
Flow Cyt, ICC/IF, IHC-Fr, WB

SpeciesHu, Ms, Rat, Ce, Cow, Zfsh

Anti-VDAC1 / Porin antibody [20B12AF2] - Mitochondrial
Loading Control (ab14734)

Other possible uses of this methodology• Monitoring movements of other proteins (e.g. AIF, Bad, Bid, Endo G, HtrA2) involved inapoptosis.

Applications
Flow Cyt, ICC/IF, IHC-P, WB• Monitoring movements of kinases and transcription factors (e.g. p53) during apoptosis.

Anti-GAPDH antibody [3E8AD9] (ab110305)• Other signaling events that involve protein re-localization.

Applications
Species
Flow Cyt, ICC/IF, IP, In-Cell ELISA, WB Hu• Separation of isoforms of enzymes, that have differential distribution, for activity assays.

ab110327
ab110315
ab110325
ab110288
ab32023ab7973ab7977ab6709ab64668
ab32371
ab114051
ab32445
ab15184ab31948ab8399ab64111ab9643SpeciesHu, Ms, Rat, Dm

160_11_MBS

Abstract