ab173187 –
Pro & Active Caspase 3
Human DUPLEX ELISA
Kit (Fluorescent)
Instructions for Use
For the quantitative simultaneous measurement of pro-caspase 3 and
p17 subunit of active caspase 3 in Human cells and tissue
homogenates.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 25/06/2013
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
4
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
5
5
5
6
6
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. STANDARDS PREPARATION
11. SAMPLE PREPARATION
12. PLATE PREPARATION
8
9
10
12
ASSAY PROCEDURE
13. ASSAY PROCEDURE
13
DATA ANALYSIS
14. CALCULATIONS
15. TYPICAL DATA
16. TYPICAL SAMPLE VALUES
17. SPECIES REACTIVITY
18. ASSAY SPECIFICITY
15
16
19
21
22
RESOURCES
19.
20.
TROUBLESHOOTING
NOTES
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24
25
1
INTRODUCTION
1.
BACKGROUND
Abcam’s Pro and Active Caspase 3 Human DUPLEX in vitro
ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for
the accurate quantitative simultaneous measurement of procaspase 3 protein and p17 subunit of active caspase 3 protein in
Human cells and tissue homogenates.
Caspase 3 is a cysteine protease involved in the activation
cascade of caspases responsible for apoptosis execution. At the
onset of apoptosis caspase 3 proteolytically cleaves poly (ADPribose) polymerase (PARP) at Asp216-Gly217 bond. Caspase 3
cleaves and activates sterol regulatory element binding proteins
(SREBPs) between the basic helix-loop-helix leucine zipper
domain and the membrane attachment domain. Caspase 3
cleaves and activates caspase-6, -7 and -9. Caspase 3 is involved
in the cleavage of huntingtin. Caspase 3 is a cytoplasmic protein
highly expressed in lung, spleen, heart, liver and kidney. Moderate
levels of caspase 3 are in brain and skeletal muscle, and low
levels in testis. Also caspase 3 is found in many cell lines, highest
expression in cells of the immune system. Caspase 3 is expressed
in an inactive pro-form (pro caspase 3). In apoptosis, the pro
caspase 3 is activated by proteolytic cleavages at Asp28-Ser29
and Asp175-Ser176 bonds catalyzed by granzyme B, caspase-6,
caspase-8, caspase-9 and caspase-10 generating two active
subunits. Thus the pro-form and the active form are useful
biomarkers of apoptosis.
Active caspase 3 is a heterotetramer that consists of two antiparallel arranged heterodimers, each one formed by a 17 kDa
(p17) and a 12 kDa (p12) subunit. Additional processing of the
propeptides is likely due to the autocatalytic activity of the
activated protease. Active heterodimers between the small subunit
of caspase-7 protease and the large subunit of caspase-3 also
occur and vice versa. Caspase 3 is S-nitrosylated on its catalytic
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2
INTRODUCTION
site cysteine in unstimulated human cell lines and denitrosylated
upon activation of the Fas apoptotic pathway, associated with an
increase in intracellular caspase activity. Fas therefore activates
caspase-3 not only by inducing the cleavage of the caspase
zymogen to its active subunits, but also by stimulating the
denitrosylation of its active site thiol.
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3
INTRODUCTION
2. ASSAY SUMMARY
Remove appropriate number
of antibody coated well
strips.
Equilibrate
all
reagents
to
room
temperature. Prepare all the
reagents,
samples,
and
standards as instructed.
Add standard or sample to
each well used. Incubate at
room temperature.
Aspirate and wash each
well. Add prepared Detector
Antibodies to each well.
Incubate
at
room
temperature.
Aspirate and wash each
well.
Add
Development
Solution to each well.
Immediately
begin
data
recording in a kinetic mode.
Alternatively record at a
user-defined time endpoint
data.
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4
GENERAL INFORMATION
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. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store all components of the kit at 4ºC immediately upon receipt
apart from the 20X Pro-Caspase3-AP Detector Antibody, which
should be stored at -20 ºC.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in section 9. Reagent Preparation.
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
2X Extraction Buffer
10X Buffer
15 mL
6 mL
4ºC
4ºC
10X Blocking Solution
10X Wash Buffer
Caspase 3 Microplate (8 x 12 antibody
coated wells)
HeLa-Vehicle Standard
HeLa-Staurosporine Standard
20X Pro-Caspase 3-AP Detector Antibody
20X Active-Caspase 3-HRP Detector
Antibody
Fluorescent Substrate Buffer
200X Fluorescent Substrate Cocktail
8000X Hydrogen Peroxide
6 mL
40 mL
4ºC
4ºC
96 Wells
4ºC
400 g
400 g
350 L
4ºC
4ºC
-20ºC
350 L
4ºC
12 mL
70 µL
50 µL
4ºC
4ºC
4ºC
Item
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5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Microplate fluorescence spectrophotometer

Method for determining protein concentration (BCA assay
recommended)

Deionized water

Multi and single channel pipettes

PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM
KCl, pH 7.3)

Tubes for standard dilution

Plate shaker for all incubation steps (optional)

Phenylmethylsulfonyl
inhibitors)
Fluoride
(PMSF)
(or
other
protease
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures

Do not use kit or components if it has exceeded the expiration date
on the kit labels

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.
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6
GENERAL INFORMATION
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers

Avoid foaming
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 multiply freeze/thaw of samples.

Incubate ELISA plates on a plate shaker during all incubation
steps (optional).

When generating positive control samples, it is advisable to
change pipette tips after each step.

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.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents to room temperature (18-25°C) prior to use.
9.1
1X Extraction Buffer
If preparing extracts from cell pellets (step 11.1) prepare
1X Extraction Buffer by adding 15 mL 2X Extraction Buffer to
15 mL nanopure water.
9.2
1X Incubation Buffer
Prepare 1X Incubation Buffer by adding 3 mL 10X Buffer and
3 mL 10X Blocking Solution to 24 mL nanopure water. Mix
gently and thoroughly.
9.3
1X Wash Buffer
Prepare 1X Wash Buffer by adding 40 mL 10X Wash Buffer
to 360 mL nanopure water. Mix gently and thoroughly.
9.4
1X Caspase 3 Detector Antibodies Cocktail
Prepare 1X Caspase 3 Detector Antibodies Cocktail by
diluting the 20X Pro-Caspase 3-AP Detector Antibody and
the 20X Active-Caspase 3-HRP Detector Antibody 20-fold
with 1X Incubation Buffer into a single cocktail immediately
prior to use. Prepare 500 µL 1X Caspase 3 Detector
Antibodies Cocktail for each 8 well strip used.
9.5
Development Solution
Prepare
Development
Solution
by
diluting
the
200X Fluorescent Substrate Cocktail and 8000X Hydrogen
Peroxide, respectively, 200-fold and 8000-fold with
Fluorescent Substrate Buffer immediately prior to use.
Prepare 1 mL Development Solution for each 8 well strip
used.
● Unused 1X Incubation Buffer should be stored at -20°C.
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8
ASSAY PREPARATION
10. STANDARDS PREPARATION
Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of positive controls for every use.
10.1 Generation of HeLa-Vehicle Standards (VEH)
10.1.1 Reconstitute HeLa-Vehicle Standard (VEH) by adding
500 µL 1X Incubation Buffer to vial. Allow to sit for 5
minutes on ice. Mix gently and thoroughly. This
800 µg/mL solution should be labeled as Standard #1
VEH (see tables below). Any remaining material of
Standard #1 VEH should be stored at -80°C.
10.1.2 Label tubes #2-8 VEH: Add 150 μL 1X Incubation
Buffer into each tube.
10.1.3 To generate Standard #2 VEH, add 150 μL Standard
#1 VEH to tube #2 VEH. Mix gently and thoroughly.
10.1.4 To generate Standard #3 VEH, add 150 μL Standard
#2 VEH to tube #3 VEH. Mix gently and thoroughly.
10.1.5 Using the table below as a guide, repeat for tubes #4
through #7 to create the rest of the required
VEH Standards.
10.1.6 Use the diluent as the zero standard tube labeled #8.
VEH-Standard Dilution Table
Standard
#
Sample to
Dilute
1-VEH
2-VEH
3-VEH
4-VEH
5-VEH
6-VEH
7-VEH
Stock Vial
#1-VEH
#2-VEH
#3-VEH
#4-VEH
#5-VEH
#6-VEH
Volume
to Dilute
(µL)
150
150
150
150
150
150
Volume of 1X
Incubation
Buffer (µL)
500
150
150
150
150
150
150
Starting
Conc.
(µg/mL)
800
400
200
100
50
25
Final
Conc.
(µg/mL)
800
400
200
100
50
25
12.5
10.2 Generation of HeLa Staurosporine Standards (STS)
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9
ASSAY PREPARATION
10.2.1 Reconstitute HeLa-Staurosporine Standard (STS) by
adding 500 µL 1X Incubation Buffer to vial. Allow to sit
for 5 minutes on ice. Mix gently and thoroughly. This
800 µg/mL should be labeled as Standard #1 STS
(see tables below). Any remaining Standard #1 STS
material should be stored at -80°C.
10.2.2 Label tubes #2-8 STS: Add 150 μL 1X Incubation
Buffer into each tube.
10.2.3 To generate Standard #2 STS, add 150 μL Standard
#1 STS to tube #2 STS. Mix gently and thoroughly.
10.2.4 To generate Standard #3 STS, add 150 μL Standard
#2 STS to tube #3 STS. Mix gently and thoroughly.
10.2.5 Using the table below as a guide, repeat for tubes #4
through #7 to create the rest of the required
STS Standards.
10.2.6 Use the diluent as the zero standard tube labeled #8.
STS-Standard Dilution Table
Standard
#
Sample to
Dilute
1-STS
2-STS
3-STS
4-STS
5-STS
6-STS
7- STS
Stock
#1-STS
#2-STS
#3-STS
#4-STS
#5-STS
#6-STS
Volume
to Dilute
(µL)
150
150
150
150
150
150
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Volume of 1X
Incubation
Buffer (µL)
500
150
150
150
150
150
150
Starting
Conc.
(µg/mL)
800
400
200
100
50
25
Final
Conc.
(µg/mL)
800
400
200
100
50
25
12.5
10
ASSAY PREPARATION
11. SAMPLE PREPARATION
TYPICAL SAMPLE DYNAMIC RANGE Typical pro-Caspase 3 working ranges
Sample Type
HeLa cells
Jurkat cells
Range (µg/mL)
12-800
3-200
Typical Active-Caspase 3 working ranges
Sample Type
HeLa cells treated for 4 hours with
1 µM staurosporine
Jurkat cells treated for 4 hours with
1 µM staurosporine
14
Range (µg/mL)
12-800
3-200
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 cell pellet at 2x107/mL in 1X Extraction
Buffer
11.1.4 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
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11
ASSAY PREPARATION
11.2 Preparation of lysates from cells in media (in-well lysis)
11.2.1 Seed cells at the same density into a multi-well plate
(e.g. 96-well plate) and treat them as desired.
11.2.2 Solubilize the cells by adding equal volume (equal to
the volume of culture media) of 2X Extraction Buffer
directly to the cells in growth media.
11.2.3 Incubate on ice for 20 minutes. If available use a
plate shaker at 300 rpm.
11.2.4 Assay samples immediately or aliquot and store at
-80°C.
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 Suspend the homogenate to 10 mg/mL in PBS.
11.3.3 Solubilize the homogenate by combining equal
volumes of 2X Extraction Buffer and the
homogenate.
11.3.4 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
●
The samples should be diluted to within the working range of the
assay in 1X Incubation Buffer.
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12
ASSAY PREPARATION
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 well plate strips should be returned to the plate packet and
stored at 4°C.
●
For each assay performed, a minimum of 2 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).
●
Well effects have not been observed with this assay. Contents of
each well can be recorded on the template sheet included in the
Resources section.
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13
ASSAY PROCEDURE
13. ASSAY PROCEDURE
●
Equilibrate all materials and prepared reagents to room
temperature prior to use.
●
It is recommended to assay both HeLa-Vehicle and HeLaStaurosporine Standards
●
It is recommended to 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,
and reseal.
13.3 Add 50 µL of each sample per well. It is recommended to
include dilution series of standard proteins (section 10), as
well as untreated sample. Also include a no material control
as a zero standard.
13.4 Cover/seal the plate and incubate for 2 hours at room
temperature.
If available use a plate shaker for all
incubation steps at 300 rpm.
13.5 Aspirate each well and wash, repeat this once more for a
total of two washes. Wash by aspirating or decanting from
wells then dispensing 300 µL 1X Wash Buffer into each well
as described above. Complete removal of liquid at each
step is essential to good performance. After the last wash,
remove the remaining buffer by aspiration or decanting.
Invert the plate and blot it against clean paper towels to
remove excess liquid.
13.6 Immediately before use prepare sufficient (500 µL/ 8 well
strip used) 1X Caspase 3 Detector Antibodies Cocktail in
Incubation Buffer (step 9.4). Add 50 µL 1X Caspase 3
Detector Antibodies Cocktail to each well used. Cover/seal
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14
ASSAY PROCEDURE
the plate and incubate for 1 hour at room temperature. If
available use a plate shaker for all incubation steps at
300 rpm.
13.7 Repeat the aspirate/wash procedure above, however,
performing a total of three washes.
13.8 Immediately before use prepare sufficient (1 mL/ 8 well strip
used) Development Solution (step 9.5). Add 100 µL
Development Solution to each empty well and immediately
begin recording in a kinetic mode in the microplate reader
prepared with the following settings:
AP
Detection
Excitation wavelength (range)
355 - 365 nm
Emission wavelength (range)
445 - 455 nm
HRP
Detection
Excitation wavelength (range)
530 - 555 nm
Emission wavelength (range)
585 - 600 nm
Time
Up to 15 min
Interval
40 – 80 sec
Shaking
Shake between reads
Alternative– In place of a kinetic reading, at a user defined,
time record the endpoint data.
13.9 Analyze the data as described below.
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15
DATA ANALYSIS
14. CALCULATIONS
●
Pro-caspase 3 is detected by alkaline phosphatase (AP).
Active caspase 3 (p17 subunit) is detected by horse radish
peroxidase (HRP).
●
For the end point readings, use relative fluorescence units (RFU).
For the kinetics readings use change of RFU/unit of time.
To interpolate the pro-caspase 3 concentrations use HeLa-Vehicle
standard curve. To interpolate the concentrations of p17 subunit of
active caspase 3 use HeLa-Staurosporine standard curve.
●
For each AP and HRP data sets, subtract average zero standard
reading from all readings. Average the duplicate readings of both
standards dilutions and plot against their concentrations. Draw the
best smooth curve through these points to construct a standard curves.
Most plate reader software or graphing software can plot these values
and curve fit. A four parameter algorithm (4PL) usually provides the
best fit, though other equations can be examined to see which
provides the most accurate (e.g. linear, semi-log, log/log, 4 parameter
logistic). Read relative proteins concentrations for unknown samples
from the standard curve plotted. Samples producing signals greater
than that of the highest standard should be further diluted and
reanalyzed, then multiplying the concentration found by the appropriate
dilution factor.
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16
DATA ANALYSIS
15. TYPICAL DATA
TYPICAL STANDARD CURVES – Data provided for demonstration
purposes only. A new standard curves must be generated for each
assay performed.
Pro-Caspase 3 Standard Curve Measurements
using HeLa-Vehicle Standard
Change RFU/sec (360/450 nm)
Conc.
(µg/mL)
1
2
3
Mean
800
8.377
8.560
8.163
8.366
400
5.715
5.727
5.737
5.726
200
3.470
3.431
3.384
3.429
100
1.949
2.010
1.967
1.975
50
1.067
1.047
1.046
1.053
25
0.571
0.555
0.546
0.557
12.5
0.301
0.301
0.282
0.295
0.0
0.100
0.035
0.041
0.059
Figure 1. Example pro-caspase 3 standard curve using HeLa-Vehicle
Standard. The HeLa-Vehicle Standard was prepared using HeLa cells
treated for 4 hours with vehicle (DMSO). Raw data values are shown in
the table. Background-subtracted data values (mean +/- SD) are
graphed.
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17
DATA ANALYSIS
Active-Caspase 3 Standard Curve Measurements
using HeLa-Staurosporine Standard
Change RFU/sec (555/595 nm)
Conc.
(µg/mL)
1
2
3
Mean
800
4.896
4.686
4.922
4.835
400
2.707
2.640
2.749
2.698
200
1.410
1.367
1.369
1.382
100
0.709
0.694
0.712
0.705
50
0.324
0.315
0.315
0.318
25
0.138
0.139
0.141
0.139
12.5
0.070
0.070
0.069
0.070
0.0
0.055
0.044
0.045
0.048
Figure 2. Example active-caspase 3 standard curve using HeLaStaurosporine Standard. The HeLa-Staurosporine Standard was
prepared using HeLa cells treated for 4 hours with 1 µM staurosporine
(ab120056). Raw data values are shown in the table. Backgroundsubtracted data values (mean +/- SD) are graphed.
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18
DATA ANALYSIS
Figure 3. Example induction of caspase 3 cleavage by staurosporine
treatment in HeLa and Jurkat cells. Cells were treated with vehicleDMSO (A and C) and 1 µM staurosporine (B and D) for 4 hours.
Extracts prepared from cell pellets were analyzed in triplicates. Raw
pro-caspase 3 signals (solid line) recorded at 360/450
(excitation/emission) nm and active-caspase 3 signals (dashed line)
recorded at 555/595 nm are shown. Dotted lines represent no material
background signal.
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19
DATA ANALYSIS
Figure 4. Example of staurosporine IC50 determination. Lysates
corresponding to 0.33x106 HeLa cells/mL were prepared by direct inwell lysis (without media removal) from cells treated for 4 hours with
variable doses of staurosporine (ab120056) in a 96-well plate. 100 µL
of the lysates were analyzed in triplicates. Background-subtracted procaspase 3 (solid line) and active-caspase 3 (dashed line) signals are
shown in left panel. Relative pro-caspase 3 (solid line) and activecaspase 3 (dashed line) concentrations interpolated from appropriate
standard curves and expressed as percent of maximal signals are
shown in right panels. Staurosporine EC50 of pro-caspase 3 was
0.9 µM. Staurosporine EC50 of active-caspase 3 was 1.1 µM.
16. TYPICAL SAMPLE VALUES
SENSITIVITY Pro-Caspase 3: Calculated minimum detectable dose = 3 µg/mL (zero
dose n = 22 + 2 standard deviations) using HeLa-Vehicle Standard.
Active-Caspase 3: Calculated minimum detectable dose = 16 µg/mL
(zero dose n = 22 + 2 standard deviations) using HeLa-Staurosporine
Standard.
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20
DATA ANALYSIS
RECOVERY –
(Sample spiking in representative sample matrices)
Pro-Caspase 3
Sample Type
50% culture media
(10FHGDMEM)
10% goat serum
50% Extraction Buffer
Average %
Recovery
Range
105
105-106
69
115
66-71
113-116
Active-Caspase 3
Sample Type
50% culture media
(10FHGDMEM)
10% goat serum
50% Extraction Buffer
Average %
Recovery
Range
103
102-104
109
102
107-11
98-104
LINEARITY OF DILUTION –
Pro-caspase 3 linearity of dilution was determined by comparing
dilution series of extracts prepared from vehicle-treated Jurkat cells to
dilution series of the HeLa-Vehicle Standard.
JurkatVehicle
Dilution
(fold)
JurkatVehicle
(µg/mL)
Interpolated
Relative Value
% Expected
Value
Undiluted
2
4
8
16
32
64
200.00
100.00
50.00
25.00
12.50
6.25
3.13
1270
634
304
151
72
37
18
100
100
96
95
91
94
89
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21
DATA ANALYSIS
Active-caspase 3 linearity of dilution was determined by comparing
dilution series of extracts prepared from staurosporine-treated Jurkat
cells to dilution series of the HeLa-Staurosporine Standard.
JurkatStaurosporine
Dilution
(fold)
Undiluted
2
4
8
16
32
64
JurkatStaurosporine
(µg/mL)
Interpolated
Relative Value
% Expected
Value
200.00
100.00
50.00
25.00
12.50
6.25
3.13
1230
566
267
127
58
27
15
100
92
87
83
76
70
77
PRECISION –
Mean of coefficient of variations within working range using appropriate
standards.
Pro-Caspase 3
n=
%CV
Intra-Assay
3
2.0
Inter-Assay
3
6.4
Active-Caspase 3
n=
%CV
Intra-Assay
3
2.1
Inter-Assay
3
11.6
17. SPECIES REACTIVITY
This kit detects pro and active caspase 3 in Human samples only.
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22
DATA ANALYSIS
18. ASSAY SPECIFICITY
Staurosporine is a protein kinase inhibitor known to induce apoptosis
involving cleavage of pro-caspase 3 into p17 and p12 subunits of
active caspase 3. Western blot analysis of the proteins
immunocaptured with the use of the Caspase 3 Microplate shows that
the microplate captures both the pro-caspase 3 and the p17 subunit of
active caspase 3 (Figure 5). As expected, with the use of the assay kit,
the staurosporine treatment reduced the levels of pro-caspase 3 about
5-fold in HeLa cells and 6-fold in Jurkat cells (Figure 6). The levels of
active caspase 3 were virtually undetectable in healthy vehicle-treated
cells (Figure 6). However, the staurosporine treatment induced
dramatic increase in the active caspase 3 levels in both cell lines
(Figure 6).
Figure 5. Demonstration of the capture antibody specificity. Cell
extracts were prepared from 4 hours vehicle-treated (lanes 1 and 3)
and 1 µM staurosporine-treated (lanes 2 and 4) Jurkat cells. Extracts
were incubated with the Caspase 3 Microplate, captured proteins were
extracted and analyzed by Western blotting using a pro+p17 caspase
3 antibody ab32351 (Lanes 3 and 4). 25% of the amounts of the
extracts used for immunocapture were also analyzed directly by the
Western blotting (lanes 1 and 2) with the same antibody. Note that the
Caspase 3 Microplate captures both the pro- and p17 subunit of
caspase 3.
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23
DATA ANALYSIS
Figure 6. Demonstration of assay specificity. Demonstration of the
assay specificity by induction of pro-caspase 3 cleavage to p17 subunit
of active caspase 3 by staurosporine treatment. Cells were treated for
4 hours with 1 µM staurosporine or drug’s vehicle (DMSO). HeLa cell
extracts (400, 200 and 100 g/mL, graphed left to right) or Jurkat cell
extracts (50, 25 and 12.5 g/mL, graphed left to right) were analyzed
using this kit. Relative pro-caspase 3 concentrations were interpolated
from HeLa-Vehicle standard curve and expressed as percent of
vehicle-treated HeLa cell. Relative active caspase 3 concentrations
were interpolated from HeLa-Staurosporine standard curve and
expressed as percent of staurosporine-treated HeLa cell. Note that (1)
staurosporine treatment reduced the pro-caspase 3 levels in both cell
lines, (2) staurosporine treatment strongly induced the active caspase
3 levels in both cell lines, (3) Jurkat cells contained approximately 6
times higher caspase 3 concentrations (per total protein mass)
compared to HeLa cells.
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24
RESOURCES
19. TROUBLESHOOTING
Problem
Poor standard
curve
Low Signal
Large CV
Low sensitivity
Cause
Solution
Inaccurate Pipetting
Check pipets
Improper standard
dilution
Prior to opening, briefly spin
the stock standard tube and
dissolve the powder
thoroughly by gentle mixing
Incubation times too
brief
Ensure sufficient incubation
times; change to overnight
standard/sample incubation
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Incubation times with
development solution
too brief
Ensure sufficient incubation
time till signal develops
Plate is insufficiently
washed
Review manual for proper
wash technique. If using a
plate washer, check all ports
for obstructions
Contaminated wash
buffer
Make fresh wash buffer
Improper storage of
the ELISA kit
Store your reconstituted
standards at -80°C, all other
assay components 4°C.
Keep substrate solution
protected from light
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25
RESOURCES
20. NOTES
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26
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RESOURCES
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