How to use plant 4-coumaric acid coenzyme A ligase (4CL) ELISA kit

Read this manual carefully before use
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This ELISA kit is based on the principle of double antibody sandwich technology to detect plant 4-coumarate coenzyme A ligase (4CL), which can only be used for research purposes, not for medical diagnosis
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Application: Used to measure the activity of plant 4-coumarate coenzyme A ligase (4CL) in plant tissues, cells and other related samples
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Working principle
This kit uses biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of plant 4-coumaric acid coenzyme A ligase (4CL) in the sample
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Plant 4-coumarate coenzyme A ligase (4CL) was added to the enzyme-labeled wells pre-coated with plant 4-coumarate coenzyme A ligase (4CL) monoclonal antibody, and incubated; after incubation, biological protein-labeled anti-4CL antibody
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It is then combined with streptavidin-HRP to form an immune complex, which is then incubated and washed to remove unbound enzymes, and then substrates A and B are added to produce blue color, which is converted into the final product under the action of acid.
of yellow
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The shade of color was positively correlated with the concentration of plant 4-coumarate-CoA ligase (4CL) in the sample
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Kit composition 

Kit composition	48 well configuration	96-well configuration	save
manual	1 serving	1 serving
Sealing film	2 pieces (48)	2 pieces (96)
sealed bag	1	1
ELISA coated plate	1×48	1×96	Store at 2-8°C
Standard product: 400ng/L	0. 5ml×1 bottle	0. 5ml×1 bottle	Store at 2-8°C
Standard Diluent	3ml×1 bottle	6ml×1 bottle	Store at 2-8°C
Streptavidin-HRP	3 ml×1 bottle	6 ml×1 bottle	Store at 2-8°C
Biotinylated anti-4CL antibody	0. 5ml×1 bottle	1 ml×1 bottle	Store at 2-8°C
Color developer liquid A	3 ml×1 bottle	6 ml×1 bottle	Store at 2-8°C
Color developer liquid B	3 ml×1 bottle	6 ml×1 bottle	Store at 2-8°C
Stop solution	3ml×1 bottle	6ml×1 bottle	Store at 2-8°C
Concentrated washing liquid	(20ml × 20 times) × 1 bottle	(20ml × 30 times) × 1 bottle	Store at 2-8°C

Reagents and equipment required but not provided

• 37°C incubator
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• Standard specification microplate reader
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• Precision pipettes and disposable tips
• distilled water,
• disposable test tube
• Absorbent paper

Precautions

• Remove the kit from 2-8°C and equilibrate at room temperature for at least 30 minutes before opening the kit
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  If the enzyme-labeled plate is not used up after opening, the slats should be stored in a sealed bag
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• A sampler should be used for each step of sample addition, and its accuracy should be checked frequently to avoid experimental errors
• Strictly follow the instructions in the instructions, and the test results must be determined by the reading of the microplate reader.
  
• To avoid cross-contamination, avoid reusing tips and sealing membranes in your hands
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• Other reagents not in use should be packaged or covered
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  Do not mix reagents from different batches
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  Use before warranty
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• Substrate B is sensitive to light, avoid prolonged exposure to light
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Plate washing method
Manual plate washing method: shake off the liquid in the ELISA plate; lay a few layers of absorbent paper on the test bench, and tap the ELISA plate downward several times; inject at least 0.
35ml of the diluted washing solution into the well, Soak for 1-2 minutes
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Repeat this process as many times as necessary
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Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after skilled use.

 
Specimen requirements
1.
Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity
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2.
The extraction should be carried out as soon as possible after the specimen collection, and the extraction should be carried out according to the relevant literature
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If the test cannot be performed immediately, the specimen can be stored at -20°C, but repeated freezing and thawing should be avoided
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operating procedure

• Dilution of the 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
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  )

200ng/L	Standard No. 5	Add 120μl of standard dilution to 120μl of double standard
100ng/L	Standard No. 4	Add 120 μl of standard No. 5 to 120 μl of standard dilution
50ng/L	Standard No. 3	Add 120μl of standard No. 4 to 120μl of standard dilution
25ng/L	Standard No. 2	Add 120μl of standard No. 3 to 120μl of standard dilution
12. 5ng/L	Standard No. 1	Add 120μl of standard No. 2 to 120μl of standard dilution

 

• Determine the number of strips required based on the number of samples to be tested plus the number of standards
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  Duplicate wells are recommended for each standard and blank well
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  Each sample is determined according to its own quantity, and those that can use duplicate holes should be made as duplicate holes as possible
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• Add sample: 1) Blank well, blank control well without sample, biotin-labeled anti-4CL antibody, streptavidin-HRP, only add chromogenic reagent A&B and stop solution, the rest of the steps are the same; 2) Standard Sample well: add 50ul of standard product, 50ul of streptavidin-HRP (biotin antibody has been integrated in the standard product, so it is not added); 3) Substitute sample well: add 40ul of sample, and then add 10ul of anti-4CL antibody to each well , Streptavidin-HRP 50ul, cover with sealing film, gently shake and mix, and incubate at 37°C for 60 minutes
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• Dosing: Dilute the 30-fold concentrated washing solution with distilled water 30-fold for later use
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• Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds, and then discard, repeat this process 5 times, and pat dry
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• Color development: Add 50ul 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 15 minutes in the dark.
   
• Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue turns to yellow at this time)
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• Determination: Zero the blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm
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  The measurement should be carried out within 10 minutes after adding the stop solution
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• Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample
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  It can also be calculated using various application software
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Summary of operating procedures:
 
Detection range: 6ng/L-200ng/L
Storage: 2-8℃
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Validity period: 6 months (2-8℃)
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