| TiterZyme® Kits
(ELISA Kits) |
|
 |
| human
Cytochrome c TiterZyme® (EIA) Kit |
| Assay Format |
Catalog # |
Kit Insert |
MSDS |
Assay Layout |
| 96 Well Kit |
900-141 |
|
|
|
|
|
| |
FOR RESEARCH PURPOSES ONLY, NOT FOR DIAGNOSTIC USE. |
 
|
-Measure concentrations in both mitochondrial & cytosolic fractions from
cell lysates
-Stable, Liquid Color-Coded Reagents
-Time to Answer: 3 Hours, 15 Minutes
-Sensitive to 6.03 pg/mL
|
Cytochrome c, ~13 kDa and 105 amino acids long, is a nuclear-encoded
component of the mitochondrial respiratory chain that is imported as an
apoenzyme into mitochondria, where it is converted to the mature form by
the addition of a heme group. It catalyzes electron transfer between
complexes III and IV of the respiratory chain, moving within the planar
surface of the inner mitochondrial membrane. In addition to Cytochrome
c's role in oxidative phosphorylation, the release of Cytochrome c from
the mitochondrial intermembrane space to the cytosol results in nuclear
apoptosis. Binding of APAF1 to Cytochrome c allows APAF1 to form a
ternary complex with and activate the initiator Procaspase-9 in the
presence of dATP. Active Caspase-9 then triggers downstream effector
caspases, beginning the death cascade apoptosis. Recent studies have
demonstrated that Cytochrome c directly microinjected in the cytoplasms
of a variety of cell types is capable of initiating apoptosis on its own,
while APAF-1 is needed as a cofactor in cell free extracts thus
emphasizing Cytochrome c's crucial role in apoptosis. In contrast,
examples of apoptosis without Cytochrome c release indicate that this
event may not be necessary in all apoptotic pathways.
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|
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1. Pipet Standards and Samples in duplicate into the wells and incubate.
2. Aspirate and wash the plate.
3. Pipet the yellow Antibody into the wells and incubate.
4. Aspirate and wash the plate.
5. Pipet the blue Conjugate into the wells and incubtate.
6. Aspirate and wash the plate.
7. Pipet Substrate into the wells and incubate.
8. Pipet Stop solution and read plate on a plate reader at 405 nm.
9. Calculate sample concentrations from the Standard Curve.
|
| |
human Cytochrome c
(900-141) |
| RANGE |
28.13 - 900 pg/mL
|
| SAMPLE SIZE |
100 µL |
| SAMPLES PER 96
WELL KIT |
40 in duplicate |
| SENSITIVITY |
6.03 pg/mL |
| PRECISION |
2.7-4.2% Intra
4.9-10.2% Inter |
SAMPLE TYPE
recommended dilution |
RIPA Cell Lysis Buffer
2 (none)
Subcellular pellet lysed in RIPA (1:2,000*)
Digitonin Cell Permeabilization Buffer (none)
Cystolic fraction lysed in Digitonin (1:300*)
|
| TIME TO ANSWER |
3 Hours, 15 Minutes
|
| SPECIES
SPECIFICITY |
human |
| *NOTE: The recommended dilution for the
cell lysate fractions is high due to the high concentration of
cells (7 million HeLa cells per mL) used. These dilutions were
necessary to get the level of Cytochrome c found in the
fractions to fall within the standard range. |
|
| Compound |
Cross Reactivity |
Compound |
Cross Reactivity |
| human Cytochrome c |
100% |
rat Cytochrome c |
6.41% |
| equine Cytochrome c |
0.92% |
canine Cytochrome c |
0.62% |
| bovine Cytochrome c |
0.61% |
|
|
| <0.03%: Bax,
Bcl-2, Bcl-XL, Caspase-9, cIAP-1, Survivin |
|
+32 (0) 16
58 90 45 
+32 (0) 16
50 90 45