Version 2 Last updated 18 November 2021

ab221832 Human
Cytocrome C
SimpleStep ELISA® Kit

For the quantitative measurement of Cytochrome C in human cell

and tissue extracts and subcellular fractions.

This product is for research use only and is not intended for
diagnostic use.

Copyright © 2017 Abcam. All rights reserved

Table of Contents

1. Overview 1
2. Protocol Summary 2
3. Precautions 3
4. Storage and Stability 3
5. Limitations 4
6. Materials Supplied 4
7. Materials Required, Not Supplied 5
8. Technical Hints 5
9. Reagent Preparation 7
10. Standard Preparation 8
11. Sample Preparation 9
12. Plate Preparation 11
13. Assay Procedure 12
14. Calculations 14
15. Typical Data 15
16. Typical Sample Values 16
17. Assay Specificity 22
18. Species Reactivity 23
19. Troubleshooting 24
20. Notes 25

Copyright © 2017 Abcam. All rights reserved

1. Overview

Cytochrome C in vitro SimpleStep ELISA® (Enzyme-Linked

Immunosorbent Assay) kit is designed for the quantitative
measurement of Cytochrome C protein in human cell and tissue
extracts and subcellular fractions.

The SimpleStep ELISA® employs an affinity tag labeled capture

antibody and a reporter conjugated detector antibody which
immunocapture the sample analyte in solution. This entire complex
(capture antibody/analyte/detector antibody) is in turn immobilized
via immunoaffinity of an anti-tag antibody coating the well. To
perform the assay, samples or standards are added to the wells,
followed by the antibody mix. After incubation, the wells are washed
to remove unbound material. TMB Development Solution is added
and during incubation is catalyzed by HRP, generating blue
coloration. This reaction is then stopped by addition of Stop Solution
completing any color change from blue to yellow. Signal is
generated proportionally to the amount of bound analyte and the
intensity is measured at 450 nm. Optionally, instead of the endpoint
reading, development of TMB can be recorded kinetically at 600
nm.

Cytochrome C is 11 kDa mitochondrial intermembrane space

electron carrier protein. The oxidized form of the cytochrome C
heme group can accept an electron from the heme group of the
cytochrome c1 subunit of cytochrome reductase. Cytochrome C
then transfers this electron to the cytochrome oxidase complex, the
final protein carrier in the mitochondrial electron-transport chain.
Cytochrome C also plays a role in apoptosis. Suppression of the anti-
apoptotic members or activation of the pro-apoptotic members of
the Bcl-2 family leads to altered mitochondrial outer membrane
permeability resulting in release of cytochrome C into the cytosol.
Binding of cytochrome C to Apaf-1 triggers the activation of
caspase-9, which then accelerates apoptosis by activating other
caspases.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 1

2. Protocol Summary

Prepare all reagents, samples, and standards as instructed

Add 50 µL standard or sample to appropriate wells

Add 50 µL Antibody Cocktail to all wells

Incubate at room temperature for 1 hour

Aspirate and wash each well three times with 350 µL 1X Wash Buffer
PT

Add 100 µL TMB Development Solution to each well and incubate

for 6 minutes.

Add 100 µL Stop Solution and read OD at 450 nm

ab221832 Human Cytochrome C SimpleStep ELISA Kit 2

3. Precautions

Please read these instructions carefully prior to beginning the assay.

 All kit components have been formulated and quality control
tested to function successfully as a kit.
 We understand that, occasionally, experimental protocols might
need to be modified to meet unique experimental
circumstances. However, we cannot guarantee the
performance of the product outside the conditions detailed in
this protocol booklet.
 Reagents should be treated as possible mutagens and should be
handled with care and disposed of properly. Please review the
Safety Datasheet (SDS) provided with the product for information
on the specific components.
 Observe good laboratory practices. Gloves, lab coat, and
protective eyewear should always be worn. Never pipet by
mouth. Do not eat, drink or smoke in the laboratory areas.
 All biological materials should be treated as potentially
hazardous and handled as such. They should be disposed of in
accordance with established safety procedures.

4. Storage and Stability

Store kit at +4°C immediately upon receipt. Kit has a storage time of
1 year from receipt, providing components have not been
reconstituted.
Refer to list of materials supplied for storage conditions of individual
components.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 3

5. Limitations

 Assay kit intended for research use only. Not for use in diagnostic
procedures.
 Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.

6. Materials Supplied

Storage
Item Quantity
Condition
Human Cytochrome C Capture Antibody 10X 600 µL +4ºC
Human Cytochrome C Detector Antibody 10X 600 µL +4ºC
Human Cytochrome C Lyophilized
2 Vials +4ºC
Recombinant Protein
Antibody Diluent CPI 6 mL +4ºC
Wash Buffer PT 10X 20 mL +4ºC
Cell Extraction Buffer PTR 5X 10 mL +4ºC
Cell Extraction Enhancer Solution 50X 1 mL +4ºC
Denaturant 500 L +4ºC
TMB Development Solution 12 mL +4ºC
Stop Solution 12 mL +4ºC
Sample Diluent NS* 12 mL +4ºC
Anti-tag coated microplate (12 x 8 well strips) 96 Wells +4ºC
Plate Seal 1 +4ºC

*Sample Diluent NS is provided but not necessary for this product.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 4

7. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to
successfully perform this assay:
 Microplate reader capable of measuring absorbance at 450 or
600 nm.
 Method for determining protein concentration (BCA assay
recommended).
 Deionized water.
 Multi- and single-channel pipettes.
 Tubes for standard dilution.
 Plate shaker for all incubation steps.
 Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease
inhibitors).

8. Technical Hints

 Samples generating values higher than the highest standard

should be further diluted in the appropriate sample dilution
buffers.
 Avoid foaming or bubbles when mixing or reconstituting
components.
 Avoid cross contamination of samples or reagents by changing
tips between sample, standard and reagent additions.
 Ensure plates are properly sealed or covered during incubation
steps.
 Complete removal of all solutions and buffers during wash steps
is necessary to minimize background.
 As a guide, typical ranges of sample concentration for
commonly used sample types are shown below in Sample
Preparation (section 11).
 All samples should be mixed thoroughly and gently.
 Avoid multiple freeze/thaw of samples.
 Incubate ELISA plates on a plate shaker during all incubation
steps.
 When generating positive control samples, it is advisable to
change pipette tips after each step.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 5

 The provided Cell Extraction Enhancer Solution 50X may
precipitate when stored at + 4ºC. To dissolve, warm briefly at +
37ºC and mix gently. The Cell Extraction Enhancer Solution 50X
can be stored at room temperature to avoid precipitation.
 To avoid high background always add samples or standards to
the well before the addition of the antibody cocktail.
 This kit is sold based on number of tests. A ‘test’ simply refers to a
single assay well. The number of wells that contain sample,
control or standard will vary by product. Review the protocol
completely to confirm this kit meets your requirements. Please
contact our Technical Support staff with any questions.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 6

9. Reagent Preparation

 Equilibrate all reagents to room temperature (18-25°C) prior to

use. The kit contains enough reagents for 96 wells. The sample
volumes below are sufficient for 48 wells (6 x 8-well strips); adjust
volumes as needed for the number of strips in your experiment.
 Prepare only as much reagent as is needed on the day of the
experiment. Capture and Detector Antibodies have only been
tested for stability in the provided 10X formulations.

9.1 1X Cell Extraction Buffer PTR:

Prepare 1X Cell Extraction Buffer PTR by diluting Cell Extraction
Buffer PTR 5X and 50X Cell Extraction Enhancer Solution to 1X
with deionized water. To make 10 mL 1X Cell Extraction Buffer
PTR combine 8 mL deionized water and 2 mL Cell Extraction
Buffer PTR 5X. Mix thoroughly and gently. If required protease
inhibitors can be added.
9.2 1X Cell Extraction Buffer PTR + Enhancer
Prepare 1X Cell Extraction Buffer PTR + Enhancer by diluting 5X
Cell Extraction Buffer PTR and 50X Cell Extraction Enhancer
Solution to 1X with deionized water. To make 10 mL 1X Cell
Extraction Buffer PTR + Enhancer combine 7.8 mL deionized
water, 2 mL 5X Cell Extraction Buffer PTR and 200 µL 50X Cell
Extraction Enhancer Solution. Mix thoroughly and gently. If
required protease inhibitors can be added.
9.3 1X Wash Buffer PT:
Prepare 1X Wash Buffer PT by diluting Wash Buffer PT 10X with
deionized water. To make 50 mL 1X Wash Buffer PT combine 5
mL Wash Buffer PT 10X with 45 mL deionized water. Mix
thoroughly and gently.
9.4 Antibody Cocktail:
Prepare Antibody Cocktail by diluting the capture and
detector antibodies in Antibody Diluent CPI. To make 3 mL of
the Antibody Cocktail combine 300 µL 10X Capture Antibody
and 300 µL 10X Detector Antibody with 2.4 mL Antibody
Diluent CPI. Mix thoroughly and gently.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 7

10. Standard Preparation

 Always prepare a fresh set of standards for every use.

 Discard working standard dilutions after use as they do not store
well.
 The following section describes the preparation of a standard
curve for duplicate measurements (recommended).

10.1 IMPORTANT: If the protein standard vial has a volume identified

on the label, reconstitute the human Cytochrome C standard
by adding that volume of 1X Cell Extraction Buffer PTR +
Enhancer indicated on the label. Alternatively, if the vial has a
mass identified, reconstitute the human Cytochrome C
standard by adding 500 µL 1X Cell Extraction Buffer PTR +
Enhancer. Hold at room temperature for 10 minutes and mix
gently. This is the 300 ng/mL Stock Standard Solution.
10.2 Label eight tubes, Standards 1– 8.
10.3 Add 225 μL 1X Cell Extraction Buffer PTR +Enhancer into tube
number 1 and 150 μL of 1X Cell Extraction Buffer PTR +
Enhancer into numbers 2-8.
10.4 Use the Stock Standard to prepare the following dilution series.
Standard #8 contains no protein and is the Blank control:

75
µL 150 150 150 150 150 150
µL µL µL µL µL µL

µ µ µ µ µ µ

300 75 37.5 18.8 9.38 4.69 2.34 1.17 0

ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL

ab221832 Human Cytochrome C SimpleStep ELISA Kit 8

11. Sample Preparation

Typical Sample Dynamic Range

Sample Type Range
Human Heart Tissue Extract 0.31 – 20 µg/mL
Human Colon Tissue Extract 1.56 – 50 µg/mL
Human Skeletal Muscle Tissue 0.78 – 25 µg/mL
Extract
HepG2 Cell Extract 1.56 – 50 µg/mL
PC-3 Cell Extract 0.78 – 50 µg/mL
THP-1 Cell Extract 3.13 – 50 µg/mL

11.1 Preparation of extracts from cell pellets:

11.1.1 Collect non-adherent cells by centrifugation or scrape to
collect adherent cells from the culture flask. Typical
centrifugation conditions for cells are 500 x g for 5 minutes at
4ºC.
11.1.2 Rinse cells twice with PBS.
11.1.3 Solubilize pellet at 2x107 cell/mL in chilled 1X Cell Extraction
Buffer PTR + Enhancer.
11.1.4 Incubate on ice for 20 minutes.
11.1.5 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.1.6 Transfer the supernatants into clean tubes and discard the
pellets.
11.1.7 At this point extract samples can be aliquoted and stored at
-80°C. The sample protein concentration in the extract may
be quantified using a protein assay.
11.1.8 To 8 volume parts of extract add 1 volume part of
Denaturant. Mix thoroughly and gently.
11.1.9 Incubate samples at room temperature for 10 minutes.
11.1.10Dilute samples 40-fold in 1X Cell Extraction Buffer PTR. Mix
thoroughly and gently.
11.1.11Dilute samples further to desired concentration in 1X Cell
Extraction Buffer PTR + Enhancer.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 9

11.2 Preparation of extracts from adherent cells by direct lysis
(alternative protocol):
11.2.1 Remove growth media and rinse adherent cells 2 times in
PBS.
11.2.2 Solubilize the cells by addition of chilled 1X Cell Extraction
Buffer PTR + Enhancer directly to the plate (use 750 µL - 1.5 mL
1X Cell Extraction Buffer PTR + Enhancer per confluent 15 cm
diameter plate).
11.2.3 Scrape the cells into a microfuge tube and incubate the
lysate on ice for 15 minutes.
11.2.4 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.2.5 Transfer the supernatants into clean tubes and discard the
pellets.
11.2.6 At this point extract samples can be aliquoted and stored at
-80°C. The sample protein concentration in the extract may
be quantified using a protein assay.
11.2.7 To 8 volume parts of extract add 1 volume part of
Denaturant. Mix thoroughly and gently.
11.2.8 Incubate samples at room temperature for 10 minutes.
11.2.9 Dilute samples 40-fold in 1X Cell Extraction Buffer PTR. Mix
thoroughly and gently.
11.2.10Dilute samples further to desired concentration in 1X Cell
Extraction Buffer PTR + Enhancer.
11.3 Preparation of extracts from tissue homogenates:
11.3.1 Tissue lysates are typically prepared by homogenization of
tissue that is first minced and thoroughly rinsed in PBS to
remove blood (dounce homogenizer recommended).
11.3.2 Homogenize 100 to 200 mg of wet tissue in 500 µL – 1 mL of
chilled 1X Cell Extraction Buffer PTR + Enhancer. For lower
amounts of tissue adjust volumes accordingly.
11.3.3 Incubate on ice for 20 minutes.
11.3.4 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.3.5 Transfer the supernatants into clean tubes and discard the
pellets.
11.3.6 At this point extract samples can be aliquoted and stored at
-80°C. The sample protein concentration in the extract may
be quantified using a protein assay.
11.3.7 To 8 volume parts of extract add 1 volume part of
Denaturant. Mix thoroughly and gently.
11.3.8 Incubate samples at room temperature for 10 minutes.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 10

11.3.9 Dilute samples 40-fold in 1X Cell Extraction Buffer PTR. Mix
thoroughly and gently.
11.3.10Dilute samples further to desired concentration in 1X Cell
Extraction Buffer PTR + Enhancer.
11.4 Subcellular fractions
11.4.1 To 8 volume parts of a fraction add 1 volume part of
Denaturant. Mix thoroughly and gently.
11.4.2 Incubate samples at room temperature for 10 minutes.
11.4.3 Dilute samples 40-fold in 1X Cell Extraction Buffer PTR. Mix
thoroughly and gently.
11.4.4 If needed, dilute samples further to desired concentration in
1X Cell Extraction Buffer PTR + Enhancer.

12. Plate Preparation

 The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.
 Unused plate strips should be immediately returned to the foil
pouch containing the desiccant pack, resealed and stored at
4°C.
 For each assay performed, a minimum of two wells must be used
as the zero control.
 For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
 Differences in well absorbance or “edge effects” have not been
observed with this assay.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 11

13. Assay Procedure

 Equilibrate all materials and prepared reagents to room

temperature prior to use.
 We recommend that you assay all standards, controls and
samples in duplicate.

13.1 Prepare all reagents, working standards, and samples as

directed in the previous sections.
13.2 Remove excess microplate strips from the plate frame, return
them to the foil pouch containing the desiccant pack, reseal
and return to 4ºC storage.
13.3 Add 50 µL of all sample or standard to appropriate wells.
13.4 Add 50 µL of the Antibody Cocktail to each well.
13.5 Seal the plate and incubate for 1 hour at room temperature
on a plate shaker set to 400 rpm.
13.6 Wash each well with 3 x 350 µL 1X Wash Buffer PT. Wash by
aspirating or decanting from wells then dispensing 350 µL 1X
Wash Buffer PT into each well. Wash Buffer PT should remain in
wells for at least 10 seconds. Complete removal of liquid at
each step is essential for good performance. After the last
wash invert the plate and tap gently against clean paper
towels to remove excess liquid.
13.7 Add 100 µL of TMB Development Solution to each well and
incubate for 6 minutes in the dark on a plate shaker set to 400
rpm.
Given variability in laboratory environmental conditions,
optimal incubation time may vary between 5 and 20 minutes.
Note: The addition of Stop Solution will change the color from
blue to yellow and enhance the signal intensity about 3X. To
avoid signal saturation, proceed to the next step before the
high concentration of the standard reaches a blue color of
O.D.600 equal to 1.0.
13.8 Add 100 µL of Stop Solution to each well. Shake plate on a
plate shaker for 1 minute to mix. Record the OD at 450 nm. This
is an endpoint reading.
Alternative to 13.7 – 13.8: Instead of the endpoint reading at
450 nm, record the development of TMB Substrate kinetically.
Immediately after addition of TMB Development Solution
begin recording the blue color development with elapsed

ab221832 Human Cytochrome C SimpleStep ELISA Kit 12

 time in the microplate reader prepared with the following
settings:

Mode Kinetic
Wavelength: 600 nm
Time: up to 20 min
Interval: 20 sec - 1 min
Shaking: Shake between readings
 Note: that an endpoint reading can also be recorded at the
completion of the kinetic read by adding 100 µL Stop Solution to
each well and recording the OD at 450 nm.
13.9 Analyze the data as described below.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 13

14. Calculations

14.1 Calculate the average absorbance value for the blank

control (zero) standards. Subtract the average blank control
standard absorbance value from all other absorbance values.
14.2 Create a standard curve by plotting the average blank
control subtracted absorbance value for each standard
concentration (y-axis) against the target protein
concentration (x-axis) of the standard. Use graphing software
to draw the best smooth curve through these points to
construct the standard curve.
 Note: Most microplate reader software or graphing software will
plot these values and fit a curve to the data. A four parameter
curve fit (4PL) is often the best choice; however, other algorithms
(e.g. linear, semi-log, log/log, 4 parameter logistic) can also be
tested to determine if it provides a better curve fit to the standard
values.
14.3 Determine the concentration of the target protein in the
sample by interpolating the blank control subtracted
absorbance values against the standard curve. Multiply the
resulting value by the appropriate sample dilution factor, if
used, to obtain the concentration of target protein in the
sample.
14.4 Samples generating absorbance values greater than that of
the highest standard should be further diluted and reanalyzed.
Similarly, samples which measure at an absorbance values less
than that of the lowest standard should be retested in a less
dilute form.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 14

15. Typical Data

Typical standard curve – data provided for demonstration purposes

only. A new standard curve must be generated for each assay
performed.

Standard Curve Measurements

Concentration O.D 450 nm Mean

(ng/mL) 1 2 O.D
0 0.070 0.067 0.069
1.17 0.146 0.131 0.138
2.34 0.207 0.203 0.205
4.69 0.327 0.299 0.313
9.38 0.557 0.514 0.535
18.8 0.993 0.949 0.971
37.5 1.781 1.765 1.773
75 3.215 3.208 3.212
Figure 1. Example of human Cytochrome C standard curve in 1X Cell
Extraction Buffer PTR + Enhancer. The Cytochrome C standard curve was
prepared as described in Section 10. Raw data values are shown in the
table. Background-subtracted data values (mean +/- SD) are graphed.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 15

16. Typical Sample Values

SENSITIVITY –
The calculated minimal detectable dose (MDD) is 1.1 ng/mL. The
MDD was determined by calculating the mean of zero standard
replicates (n=25) and adding 2 standard deviations then
extrapolating the corresponding concentration.

RECOVERY –
Three concentrations of recombinant human Cytochrome C protein
were spiked in duplicate to the indicated biological matrix to
evaluate signal recovery in the working range of the assay.
Average %
Sample Type Range (%)
Recovery

5 µg/mL Human Heart Tissue 104 101 - 106

Extract
25 µg/mL HepG2 Cell Extract 107 105 - 110

ab221832 Human Cytochrome C SimpleStep ELISA Kit 16

Linearity of Dilution
Linearity of dilution is determined based on interpolated values from
the standard curve. Linearity of dilution defines a sample
concentration interval in which interpolated target concentrations
are directly proportional to sample dilution.

Native Cytochrome C was measured in the following biological

samples in a 2-fold dilution series. Sample dilutions are made in
1X Cell Extraction Buffer PTR + Enhancer.

25 µg/mL
20 µg/mL 50 µg/mL
Human
Human Human
Dilution Skeletal
Interpolated value Heart Colon
Factor Muscle
Tissue Tissue
Tissue
Extract Extract
Extract
ng/mL 33.73 37.27 45.71
Undiluted
% Expected value 100 100 100

ng/mL 17.13 15.80 20.92

2
% Expected value 102 85 92

ng/mL 8.739 7.914 9.779

4
% Expected value 104 85 86

ng/mL 4.572 3.940 5.184

8
% Expected value 108 85 91

ng/mL 2.345 1.979 2.967

16
% Expected value 111 85 104

ab221832 Human Cytochrome C SimpleStep ELISA Kit 17

Native Cytochrome C was measured in the following biological
samples in a 2-fold dilution series. Sample dilutions are made in 1X
Cell Extraction Buffer PTR + Enhancer.
50
50 µg/mL 50 µg/mL
µg/mL
Dilution HepG2 THP-1
Interpolated value PC-3
Factor Cell Cell
Cell
Extract Extract
Extract
ng/mL 46.64 83.47 18.79
Undiluted
% Expected value 100 100 100

ng/mL 22.69 41.61 9.502

2
% Expected value 97 100 101

ng/mL 11.65 20.51 5.035

4
% Expected value 100 98 107

ng/mL 5.755 9.963 2.477

8
% Expected value 99 95 105

ng/mL 2.719 4.859 1.081

16
% Expected value 93 93 92

PRECISION –
Mean coefficient of variations of interpolated values from three
concentrations of human heart tissue extract within the working
range of the assay.
Intra- Inter-
Assay Assay

n= 3 5
CV(%) 2.8 6.3

ab221832 Human Cytochrome C SimpleStep ELISA Kit 18

Figure 2. Interpolated concentrations of native Cytochrome C in human
heart tissue extract based on a 20 μg/mL extract load, colon tissue extract
based on a 50 μg/mL extract load, and skeletal muscle tissue extract based
on a 25 μg/mL extract load. The concentrations of Cytochrome C were
measured in duplicate and interpolated from the Cytochrome C standard
curve and corrected for sample dilution. The interpolated dilution factor
corrected values are plotted (mean +/- SD, n=2). The mean Cytochrome C
concentration was determined to be 35.41 ng/mL in heart tissue extract,
32.74 ng/mL in colon tissue extract, and 43.12 ng/mL in skeletal muscle tissue
extract.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 19

Figure 3. Interpolated concentrations of native Cytochrome C in HepG2 cell
extract, PC-3 cell extract, and THP-1 cell extract samples based on a
50 μg/mL extract load. The concentrations of Cytochrome C were
measured in duplicate and interpolated from the Cytochrome C standard
curve and corrected for sample dilution. The interpolated dilution factor
corrected values are plotted (mean +/- SD, n=2). The mean Cytochrome C
concentration was determined to be 45.64 ng/mL in HepG2 cell extract,
81.23 ng/mL in PC-3 cell extract, and 19.01 ng/mL in THP-1 cell extract.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 20

Figure 4. Interpolated concentrations of native Cytochrome C in human
extract samples. The concentrations of Cytochrome C were measured in
three different dilutions in duplicate and interpolated from the Cytochrome
C standard curve and corrected for sample dilution. The interpolated
dilution factor corrected values are plotted in ng of Cytochrome C per mg
of extract (mean +/- SD, n=3). Cytochrome C concentration was
determined to be 1716 ng/mg heart tissue extract, 670.1 ng/mg in colon
tissue extract, 1689 ng/mg in skeletal muscle tissue extract, 924.2 ng/mg in
HepG2 cell extract, 1658 ng/mg in PC-3 cell extract, and 386.2 ng/mg in
THP-1 cell extract samples.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 21

17. Assay Specificity

This kit recognizes both native and recombinant human Cytochrome

C protein in cell and tissue extract, and subcellular fraction samples
only.

Figure 5. Comparison Cytochrome C distribution in subcellular fractions

derived from 3.7x103 HeLa cells and whole cells cultured in the presence
(treated) or absence (untreated) of 1 µM staurosporine for 4 hours. Cells
were collected directly after treatment and subcellular fractions were
prepared using a cell fractionation kit (ab109719). Fractions were processed
as described in section 11.10. and assayed. The concentrations of
Cytochrome C were measured in three different dilutions of the fraction
samples in duplicates and interpolated from the Cytochrome C standard
curve. The interpolated values are plotted (mean +/- SD, n=3). The mean
Cytochrome C concentration was determined be 171.4 ng/mL in the
treated cytosol fraction, 242.8 in the treated mitochondrial fraction, 562.0 in
the treated whole cell sample, 407.2 ng/mL in the untreated mitochondrial
fraction, and 558.9 ng/mL in the untreated whole cell sample. Cytochrome
C was not detectable in the untreated cytosol fraction and in both nuclear
fractions.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 22

18. Species Reactivity

This kit recognizes human Cytochrome C protein.

Other species reactivity was determined by measuring a 20 g/mL

extract load of human and rat heart tissue extract samples,
interpolating the protein concentrations from the human standard
curve, and expressing the interpolated concentrations as a
percentage of the protein concentration in the human heart tissue
extract. Cross-reactivity was determined to be 100% in rat heart
extract. Due to 100% amino acids sequence identity of rat and
mouse Cytochrome C, the same cross-reactivity can be assumed for
mouse Cytochrome C.

Please contact our Technical Support team for more information.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 23

19. Troubleshooting

Problem Reason Solution

Difficulty Prepare 1X Cell Extraction Buffer
Genomic DNA
pipetting lysate; PTR (without enhancer). Add
solubilized
viscous lysate. enhancer to lysate after extraction.
Inaccurate
Check pipettes
Pipetting
Poor standard Prior to opening, briefly spin the
curve Improper standard stock standard tube and dissolve
dilution the powder thoroughly by gentle
mixing
Ensure sufficient incubation times;
Incubation times
increase to 2 or 3 hour
too brief
standard/sample incubation
Inadequate
Check pipettes and ensure correct
Low Signal reagent volumes or
preparation
improper dilution
Ensure sufficient incubation time
Incubation times
until blue color develops prior
with TMB too brief
addition of Stop solution
Review manual for proper wash
Plate is insufficiently
technique. If using a plate washer,
washed
Large CV check all ports for obstructions.
Contaminated
Prepare fresh wash buffer
wash buffer
Store your reconstituted standards
at -80°C, all other assay
Improper storage of
Low sensitivity components 4°C. Keep TMB
the ELISA kit
Development Solution protected
from light.
Precipitation and/or
Precipitate in coagulation of Precipitate can be removed by
Diluent components within gently warming the Diluent to 37ºC.
the Diluent.

ab221832 Human Cytochrome C SimpleStep ELISA Kit 24

20. Notes

ab221832 Human Cytochrome C SimpleStep ELISA Kit 25

Technical Support
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registered trademark. All information / detail is correct at time of going to
print.

For all technical or commercial enquiries please go to:

www.abcam.com/contactus
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www.abcam.co.jp/contactus (Japan)

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