Rat 6-Keto-PGF1α
FOR RESEARCH USE ONLY
Assay range：3ng/mL –80ng/mL 96 determinations
Purpose
This kit allows for the determination of 6-Keto-PGF1α concentrations in Rat serum,
cell culture supernates ａnd other biological fluids
Principle of the assay
The kit assay Rat 6-Keto-PGF1α level in the sample，use Purified Rat 6-Keto-PGF1α
antibody to coat microtiter plate wells, make solid-phase antibody, then add 6-Keto-PGF1α to
wells, Combined 6-Keto-PGF1α antibody which With HRP labeled,become antibody - antigen -
enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB
substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition
of a sulphuric acid solution ａnd the color change is measured spectrophotometrically at a
wavelength of 450 nm. The concentration of Rat 6-Keto-PGF1α in the samples is then
determined by comparing the O.D. of the samples to the standard curve.
Materials provided with the kit
1 wash solution 20ml×1bottle 7 S Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8
Standard（160
ng/mL）
0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml×1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
Specimen requirements
2
1. extract as soon as possible after Specimen collection,and according to the relevant
literature, ａnd should be experiment as soon as possible after the extraction. If it can’t,
specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1. Dilute ａnd add sample:Dilute Original density Standard as follow table:
80ng/mL 5 Standard 150μl Original density Standard+150μl Standard diluent
40ng/mL 4 Standard 150μl 5 Standard+150μl Standard diluent
20ng/mL 3 Standard 150μl 4 Standard+150μl Standard diluent
10ng/mL 2 Standard 150μl 3 Standard +150μl Standard diluent
5ng/mL 1 Standard 150μl 2 Standard +150μl Standard diluent
2.add sample：Set blank wells separately (blank comparison wells don’t add sample and
HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample
dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is
5-fold), add sample to wells , don’t touch the well wall as far as possible, ａnd Gently mix.
3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled
water ａnd reserve.
5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer
to every well, still for 30s then drain, repeat 5 times, dry by pat.
6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul ａnd Chromogen Solution B 50ul to each well, evade
the light preservation for 10min at 37℃
10.S the reaction：Add S Solution50μl to each well, S the reaction(the blue color
change to yellow color).
11.assay：take blank well as zero , Read absorbance at 450nm after Adding S Solution and
3
within 15min.
Steps description
Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37℃.
Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.
Wash 5 times,Add Chromogen Solution A ａnd B, incubate for 10 min at 37℃.
Add S Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
Take the standard density as the horizontal, the OD value for the vertical ,draw the
standard curve on graph paper, Find out the corresponding density according to the sample
OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line
4
regression equation of the standard curve with the standard density ａnd the OD value ,with the
sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,
the result is the sample actual density.
Important notes
1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in
the room temperature, ELISA plates coated if has not use up after opened, the plate should
be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve
when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the
experimental error. add sample within 5 min, if the number of sample is much , recommend
to use Volley .
4. if the testing material content is excessively higher (The sample OD is bigger than the first
standard well ),please dilute Sample (n-fold), Please diluente ａnd multiplied by the dilution
factor.（×n×5）.
5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination must take the
microtiter plate reader as a standard.
8. All samples, washing buffer ａnd each kind of reject should according to infective material
process.
9. Do not mix reagents with those from other lots.
Storage ａnd validity
1．Storage： 2-8℃.
2．validity： six months