Technical Manual for RuBisCO ELISA Detection Kit for Plant 1,5-diphosphate ribulose Carboxylase

BS-4938 Plant 1,5-diphosphate ribulose carboxylase (RuBisCO) ELISA kit 96T/48T

Product Overview

This kit is used for in vitro quantitative detection of the activity of ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO) in plant tissues, cells, and related liquid samples. This enzyme is a key enzyme in the Calvin cycle of plant photosynthesis, and its activity directly affects the photosynthetic rate and photorespiration process. This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA), which has high sensitivity and specificity.

Detection Principle

The reagent kit adopts a double antibody one-step sandwich method:

Pre packaged microplate with anti RuBisCO monoclonal antibody captures the target enzyme in the sample

Formation of 'antibody antigen enzyme labeled antibody' complex by adding HRP labeled detection antibody

After clean washing, add TMB chromogenic substrate

After the reaction is terminated, the enzyme-linked immunosorbent assay (ELISA) is used to measure the absorbance (OD value) at a wavelength of 450nm

The concentration of RuBisCO in the sample is positively correlated with the OD value

Composition of reagent kit

Storage conditions for component specifications

Pre packaged microporous plate with 12 × 8 holes at 4 ℃

Standard product (6 concentration gradients) 0.3mL x 6 tubes -20 ℃

Enzyme labeled antibody 15mL at 4 ℃

20 x 50mL washing buffer at room temperature

TMB color solution A/B 15mL each, 4 ℃, away from light

Termination solution 15mL at room temperature

Sample diluent 30mL at 4 ℃

sample processing

Organizational samples

Weigh 0.1g of tissue and add 1mL of extract to the ice bath homogenate

Centrifuge 10000g at 4 ℃ for 10 minutes

Immediately detect the supernatant or store it at -80 ℃ (avoid repeated freezing and thawing)

Cell samples

Collect cell sediment and add extraction solution for lysis

Centrifuge 10000g at 4 ℃ for 10 minutes

Take the supernatant for detection

Serum/liquid samples

Leave at room temperature for 2 hours or overnight at 4 ℃

Centrifuge 1000g for 20 minutes

Take the supernatant for detection

Attention:

Prevent sample hemolysis

NaN3 inhibits HRP activity

Fresh samples are prioritized, and frozen samples should avoid repeated freeze-thaw cycles

operating steps

Preparation of reagents: All reagents should be equilibrated to room temperature (20-25 ℃) before use

Sample addition:

Standard well: 50 μ L of standard samples of different concentrations

Sample well: 50 μ L of sample to be tested

Blank hole: not added

Incubation: Add 100 μ L enzyme labeled antibody to each well and react at 37 ℃ in the dark for 60 minutes

Washing: Add 350 μ L of washing solution to each well, let it stand for 1 minute, shake dry, repeat 5 times

Color development: Add 50 μ L of substrates A and B to each well, and react at 37 ℃ in the dark for 15 minutes

Termination: Add 50 μ L of termination solution to each well

Measurement: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the 450nm OD value within 15 minutes

Result Calculation

Draw a standard curve with standard concentration as the horizontal axis and OD value as the vertical axis

Determine the concentration on the standard curve based on the sample OD value

Sample concentration=measured concentration x dilution factor (if any)

performance metrics

Detection range: 18-650 U/L

Sensitivity: ≤ 5 U/L

Intra batch coefficient of variation:<10%

Inter batch coefficient of variation:<15%

Precautions

The reagent kit is valid for 6 months and stored at 2-8 ℃

Different batch numbers of reagents cannot be mixed

Color developing solution stored away from light

Unclean washing can lead to false positives

The termination liquid contains corrosive substances, and protection is required during operation

All waste shall be treated as biohazard

Required instruments

ELISA reader (450nm filter)

Precision pipette (0.5-1000 μ L)

37 ℃ constant temperature box

Washing machine (optional)

Vortex mixer

Note: For scientific research purposes, the above information is for reference only and is not intended as experimental evidence. Please consult the technical teacher or brand for specific information.

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