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技术文章
大鼠（Rat）磷酸化 p38 丝裂原活化蛋白激酶 （P-P38MAPK） ELISA 检测试剂盒行业应用
点击次数：1336 发布时间：2021/5/10 17:01:01
Intended use
This P-P38MAPK ELISA kit is intended Laboratory for Research use only ａnd
is
not for use in diagnostic or therapeutic procedures.The S Solution changes the
color from blue to yellow ａnd the intensity of the color is measured at 450 nm using
a spectrophotometer. In order to measure the concentration of P-P38MAPK in the
sample, this P-P38MAPK ELISA
Kit includes a set of calibration
standards. The
calibration standards
are assayed
at the
same time
as the samples
and allow the
operator to produce
a standard
curve
of Optical
Density versus P-P38MAPK
concentration. The concentration of P-P38MAPK in the samples is then determined
by comparing the O.D. of the samples to the standard curve.
Sample collection ａnd storages
Serum - Use a serum separator tube ａnd allow samples
to clot for 30 minutes
before centrifugation for 10 minutes at approximately 3000×g. Remove serum ａnd
assay immediately or aliquot ａnd store samples at -20℃ or -80℃.Avoid repeated
freeze-thaw cycles
Plasma - Collect plasma
using EDTA or heparin
as an anticoagulant. Centrifuge
samples for 30 minutes at
3000×g at 2-8℃ within
30 minutes of collection. Store
samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates ａnd other biological fluids - Remove particulates
by centrifugation ａnd assay immediately or aliquot ａnd store samples at -20℃or
-80℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequately ａnd no hemolysis or
granule was allowed.
Materials required but not supplied
1.
Standard microplate reader(450nm)
2.
Precision pipettes ａnd Disposable pipette tips.
3.
37 ℃ incubator
Precautions
1. Do not substitute reagents from one kit to another. Standard, conjugate ａnd
microplates are matched for optimal performance. Use only the reagents supplied by
manufacturer.
2. Do not remove microplate from the storage bag until needed. Unused strips
should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.Remove all kit reagents from refrigerator ａnd allow them to reach room temperature
( 20-25°C)
Materials supplied
Name
96 determinations
48 determinations
Microelisa stripplate
12*8strips
12*4strips
Standard
0.3ml*6tubes
0.3ml*6tubes
Sample Diluent
6.0ml
3.0ml
HRP-Conjugate reagent
10.0ml
5.0ml
20X Wash solution
25ml
15ml
Chromogen Solution A
6.0ml
3.0ml
Chromogen Solution B
6.0ml
3.0ml
S Solution
6.0ml
3.0ml
Closure plate membrane
2
2
User manual
1
1
Sealed bags
1
1
Note: Standard (S0 → S5)
concentration was followed by:0,50,100,200,400,800
pg/ml
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1.
Prepare all re a g e n ts before starting assay procedure. It is recommended that
all
Standards ａnd Samples be added in duplicate to the Microelisa Stripplate.
2.
Add standard: Set Standard wells, testing sample wells. Add standard 50μl to
standard well.
3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing
sample well; Blank well doesn’t add anyting.
4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip
and incubate for 60 minutes at 37°C.
5. Aspirate each well ａnd wash, repeating the process four times for a total of five
washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle,
manifold
dispenser or autowasher. Complete
removal of liquid
at each step is
essential
to good performance. After the last
wash, remove
any
remaining Wash
Solution by aspirating or decanting. Invert the plate ａnd blot
it against clean paper
towels.
6. Add chromogen solution A 50μl ａnd chromogen solution B 50μl to each well.
Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl S Solution to each well. The color in the wells should change
from blue to yellow. If the color in the wells is green or the color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
8.
Read the Optical Density (O.D.) at 450
nm using a microtiter plate reader
within 15 minutes.
Calculation of results
1. This standard curve is used to determine
the amount in an unknown sample.The standard curve
is generated by
plotting the average
O.D. (450
nm)
obtained for each of
the six standard
concentrations on the
vertical (Y)
axis
versus the corresponding concentration on the horizontal (X) axis.
2.
First, calculate the mean O.D. value for each standard
and sample. All O.D.
values, are subtracted by the mean value of the zero
standard
before result
interpretation.
Construct the
standard curve using graph paper
or statistical
software.
3.
To determine
the amount in
each sample, first locate the O.D.
value on the
Y-axis ａnd extend a horizontal line
to the standard
curve. At
the point of
intersection, draw a vertical line to
the X-axis ａnd
read the corresponding
concentration.
4.
Any variation in operator, pipetting ａnd washing technique, incubation time or
temperature, ａnd kit age can cause variation in result. Each user should obtain
their own standard curve.
5.
The sensitivity by this assay is 1.0 pg/ml
6.
Standard curve
Storage：
2-8℃.
validity：
six months.
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR
DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
ENTIRE PROCEDURE BEFORE BEGINNING!
原创作者：上海隆诚生物科技有限公司
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