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The composition and operation steps of plant ribulose 1,5-diphosphate carboxylase (RubisCO) Experimental principle This kit uses the double antibody sandwich method to determine the level of plant ribulose 1,5-diphosphate carboxylase (RubisCO) in specimens
.
Coat the microplate with purified plant 1,5-diphosphate ribulose carboxylase (RubisCO) antibody to make a solid-phase antibody, and add plant 1,5-diphosphate nucleus to the microwells coated with monoclonal antibody in turn Keto carboxylase (RubisCO), then combined with HRP-labeled 1,5-diphosphate ribulose carboxylase (RubisCO) antibody to form an antibody-antigen-enzyme-labeled antibody complex, which was thoroughly washed and then added to the substrate TMB color
.
TMB is converted to blue under the catalysis of HRP enzyme and to the final yellow under the action of acid
.
The shade of color was positively correlated with the plant ribulose 1,5-bisphosphate carboxylase (RubisCO) in the samples
.
The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of plant ribulose 1,5-diphosphate carboxylase (RubisCO) in the samples was calculated from the standard curve
.
Kit composition: 1 30-fold concentrated washing solution 20ml×1 bottle 7 Stop solution 6ml×1 bottle 2 ELISA reagent 6ml×1 bottle 8 Standard substance (300ng/L) 0.
5ml×1 bottle 3 ELISA plate 12 wells ×8 strips 9 Standard dilution solution 1.
5ml × 1 bottle 4 Sample dilution solution 6ml × 1 bottle 10 Instructions 1 portion 5 Color developer solution A 6ml × 1 bottle 11 Sealing film 2 sheets 6 Color developer solution B 6ml × 1 / Bottle 12 sealed bags 1 plant 1,5-diphosphate ribulose carboxylase (RubisCO) composition and operation steps Specimen requirements 1.
Extract as soon as possible after specimen collection, extraction is carried out according to relevant literature, and experiments should be carried out as soon as possible after extraction
.
If the test cannot be performed immediately, the specimen can be stored at -20°C, but repeated freezing and thawing should be avoided.
2.
Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP)
.
Operation steps 1.
Dilution of standard: This kit provides a standard of original times, and the user can dilute it in a small test tube according to the following chart
.
150ng/L No.
5 standard 150μl double standard added 150μl standard dilution 75ng/L No.
4 standard 150μl No.
5 standard added 150μl standard dilution 37.
5ng/L No.
3 standard 150μl No.
4 Add 150μl of Standard Diluent 18.
75 ng/L to Standard 2.
Add 150μl of Standard No.
3 to 150μl of Standard Diluent 9.
375 ng/L No.
1 Standard 150μl of No.
2 Standard Add 150μl of Standard Diluent 2.
Add sample: set up blank wells (the blank control wells do not add sample and enzyme labeling reagents, and the other steps are the same), standard wells, and sample wells to be tested
.
Accurately add 50 μl of standard sample on the ELISA-coated plate, first add 40 μl of sample diluent to the well of the sample to be tested, and then add 10 μl 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, try not to touch the wall of the well, and shake gently to mix
.
3.
Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes
.
4.
Preparation: Dilute the 30-fold concentrated washing solution with distilled water 30-fold for later use.
5.
Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, and let it stand for 30 seconds before discarding.
, repeat 5 times, pat dry
.
6.
Add enzyme: Add 50μl of enzyme labeling reagent to each well, except for blank wells
.
7.
Incubation: The operation is the same as 3
.
8.
Washing: The operation is the same as 5
.
9.
Color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37°C for 10 minutes in the dark.
10.
Termination: add 50 μl of stop solution to each well to terminate the reaction ( At this point, the blue turns to yellow)
.
11.
Determination: Zero the blank well, and measure the absorbance (OD value) of each well in sequence at 450nm wavelength
.
The measurement should be carried out within 15 minutes after adding the stop solution
.
.
Coat the microplate with purified plant 1,5-diphosphate ribulose carboxylase (RubisCO) antibody to make a solid-phase antibody, and add plant 1,5-diphosphate nucleus to the microwells coated with monoclonal antibody in turn Keto carboxylase (RubisCO), then combined with HRP-labeled 1,5-diphosphate ribulose carboxylase (RubisCO) antibody to form an antibody-antigen-enzyme-labeled antibody complex, which was thoroughly washed and then added to the substrate TMB color
.
TMB is converted to blue under the catalysis of HRP enzyme and to the final yellow under the action of acid
.
The shade of color was positively correlated with the plant ribulose 1,5-bisphosphate carboxylase (RubisCO) in the samples
.
The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of plant ribulose 1,5-diphosphate carboxylase (RubisCO) in the samples was calculated from the standard curve
.
Kit composition: 1 30-fold concentrated washing solution 20ml×1 bottle 7 Stop solution 6ml×1 bottle 2 ELISA reagent 6ml×1 bottle 8 Standard substance (300ng/L) 0.
5ml×1 bottle 3 ELISA plate 12 wells ×8 strips 9 Standard dilution solution 1.
5ml × 1 bottle 4 Sample dilution solution 6ml × 1 bottle 10 Instructions 1 portion 5 Color developer solution A 6ml × 1 bottle 11 Sealing film 2 sheets 6 Color developer solution B 6ml × 1 / Bottle 12 sealed bags 1 plant 1,5-diphosphate ribulose carboxylase (RubisCO) composition and operation steps Specimen requirements 1.
Extract as soon as possible after specimen collection, extraction is carried out according to relevant literature, and experiments should be carried out as soon as possible after extraction
.
If the test cannot be performed immediately, the specimen can be stored at -20°C, but repeated freezing and thawing should be avoided.
2.
Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP)
.
Operation steps 1.
Dilution of standard: This kit provides a standard of original times, and the user can dilute it in a small test tube according to the following chart
.
150ng/L No.
5 standard 150μl double standard added 150μl standard dilution 75ng/L No.
4 standard 150μl No.
5 standard added 150μl standard dilution 37.
5ng/L No.
3 standard 150μl No.
4 Add 150μl of Standard Diluent 18.
75 ng/L to Standard 2.
Add 150μl of Standard No.
3 to 150μl of Standard Diluent 9.
375 ng/L No.
1 Standard 150μl of No.
2 Standard Add 150μl of Standard Diluent 2.
Add sample: set up blank wells (the blank control wells do not add sample and enzyme labeling reagents, and the other steps are the same), standard wells, and sample wells to be tested
.
Accurately add 50 μl of standard sample on the ELISA-coated plate, first add 40 μl of sample diluent to the well of the sample to be tested, and then add 10 μl 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, try not to touch the wall of the well, and shake gently to mix
.
3.
Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes
.
4.
Preparation: Dilute the 30-fold concentrated washing solution with distilled water 30-fold for later use.
5.
Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, and let it stand for 30 seconds before discarding.
, repeat 5 times, pat dry
.
6.
Add enzyme: Add 50μl of enzyme labeling reagent to each well, except for blank wells
.
7.
Incubation: The operation is the same as 3
.
8.
Washing: The operation is the same as 5
.
9.
Color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37°C for 10 minutes in the dark.
10.
Termination: add 50 μl of stop solution to each well to terminate the reaction ( At this point, the blue turns to yellow)
.
11.
Determination: Zero the blank well, and measure the absorbance (OD value) of each well in sequence at 450nm wavelength
.
The measurement should be carried out within 15 minutes after adding the stop solution
.
