Shrimp glutamate decarboxylase (GAD) Elisa kit instructions
This kit is for research use only.
Detection range: 96T
6U / L -200 U / L
purpose of usage:
This kit is used to determine the activity of glutamate decarboxylase (GAD) in shrimp serum, plasma and related liquid samples.
Experimental principle
Shrimp glutamate decarboxylase (GAD) Elisa kit instructions This kit uses the double antibody sandwich method to determine the level of shrimp glutamate decarboxylase (GAD) in the specimen. With purified shrimp
Amine decarboxylase (GAD) antibody is coated on the microplate to make a solid phase antibody, and glutamine is added to the monoclonal antibody-coated microwells in turn
Acid decarboxylase (GAD), then combined with HRP-labeled glutamate decarboxylase (GAD) antibody to form antibody-antigen-enzyme label
Antibody complex, after thorough washing, add substrate TMB to develop color. TMB turns into blue under the catalysis of HRP enzyme, and
Under the action of acid, it turns into the final yellow. The color depth is positively correlated with glutamate decarboxylase (GAD) in the sample.
Measure the absorbance (OD value) at 450nm with a microplate reader, and calculate the shrimp glutamate decarboxylase in the sample by the standard curve
(GAD) active concentration.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (400U / L) 0.5ml × 1 bottle
3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 2 sealing film
6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag
Specimen requirements
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If not
Perform the test immediately. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard products: this kit provides one original standard product, the user can dilute in a small test tube according to the following chart
release.
200 U / L No. 5 standard 150μl original standard added 150μl standard dilution
100 U / L No. 4 standard 150 μl No. 5 standard added 150 μl standard dilution
50 U / L No. 3 standard 150 μl No. 4 standard added 150 μl standard dilution
25 U / L No. 2 standard 150μl No. 3 standard added 150μl standard diluent
12.5 U / L No. 1 standard 150μl No. 2 standard added 150μl standard diluent
2. Add sample: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard well
Sample hole to be tested. Add 50μl of the standard on the enzyme-coated plate accurately, and add 40μl of sample diluent to the sample well.
Then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microtiter plate
Do not touch the wall of the hole, shake gently to mix.
3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
10 minutes.
10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated
Within 15 minutes after the solution.
Summary of operating procedures:
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample
The corresponding concentration of OD value is found by the standard curve; multiplied by the dilution factor; or the standard concentration and OD value are used to calculate the standard
The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor,
This is the actual concentration of the sample.
Precautions
1. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before it can be used.
After use, the slats should be stored in sealed bags.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the results will not be affected during washing.
3. The sample adder should be used in each step of sample addition, and its accuracy should be regularly checked to avoid test errors. One sample loading time is best
Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples.
4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole. If the content of the test substance in the specimen is too high (sample OD value
Is greater than the OD value of the first well of the standard product), please dilute it with a certain multiple (n times) of the sample diluent before measuring.
When calculating, please multiply the total dilution factor (× n × 5).
5. The sealing film is limited to one-time use to avoid cross contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader.
8. All samples, washing liquid and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
Human Ornithine decarboxylase (ODC)
FOR RESEARCH USE ONLY
Assay range: 10 U / L -320 U / L 96 determinations
Purpose
For the quantitative in vitro determination of ODC concentrations in Human serum,
cell culture supernates and other biological fluids
Principle of the assay
The kit assay Human ODC level in the sample, use Purified Human ODC antibody to
coat microtiter plate wells, make solid-phase antibody, then add ODC to wells, Combined ODC
antibody which With HRP labeled goat anti-Human 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 Human ODC in the samples is then determined by
comparing the OD of the samples to the standard curve.
Materials provided with the kit
1 wash solution 20ml × 1bottle 7 Stopp Solution 6ml × 1 bottle
2 HRP-Conjugate reagent 6ml × 1 bottle 8 Standard (640U / L) 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
RD
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:
320U / L 5 Standard 150μl Original density Standard + 150μl Standard diluent
160U / L 4 Standard 150μl 5 Standard + 150μl Standard diluent
80U / L 3 Standard 150μl 4 Standard + 150μl Standard diluent
40U / L 2 Standard 150μl 3 Standard + 150μl Standard diluent
20U / L 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 to each well, evade the
light preservation for 15 min at 37 ℃
10.Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction (the blue color
change to yellow color).
11.assay: take blank well as zero, Read absorbance at 450nm after Adding Stop Solution and
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 30 min at 37 ℃.
Add Stopp 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
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 shrimp glutamate decarboxylase (GAD) Elisa kit instructions
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