LIM Domain Binding 1 Protein ELISA Kit (LIM Domain Binding 1) ELISA Kit
- Detection Method
- Method Type
- Sandwich ELISA
- Detection Range
- 0.15 ng/mL - 10 ng/mL
- Minimum Detection Limit
- 0.15 ng/mL
- The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of LDB1 in Tissue Homogenate,Biological Fluids
- Sample Type
- Tissue Homogenate
- Analytical Method
This assay has high sensitivity and excellent specificity for detection of LIM Domain Binding Protein 1 (LDB1).
No significant cross-reactivity or interference between LIM Domain Binding Protein 1 (LDB1) and analogues was observed.
- Cross-Reactivity (Details)
- 0.061 ng/mL
- Pre-coated, ready to use 96-well strip plate
- Plate sealer for 96 wells
- Standard Diluent
- Assay Diluent A
- Assay Diluent B
- Stop Solution
- Detection Reagent A
- Detection Reagent B
- TMB Substrate
- Wash Buffer (30 x concentrate)
- Instruction manual
- Material not included
- Microplate reader with 450 nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes for diluting samples.
- Deionized or distilled water.
- Absorbent paper for blotting the microtiter plate.
- Container for Wash Solution
- Application Notes
Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.
Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.
Information on antibodies:
The provided antibodies and their host vary in different kits.
- Sample Volume
- 100 μL
- Assay Time
- 3 h
- The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to LIM Domain Binding Protein 1 (LDB1). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to LIM Domain Binding Protein 1 (LDB1). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain LIM Domain Binding Protein 1 (LDB1), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of LIM Domain Binding Protein 1 (LDB1) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
- Reagent Preparation
- Bring all kit components and samples to room temperature (18-25°C) before use.
- Making serial dilution in the wells directly is not permitted.
- The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
- Contaminated water or container for reagent preparation will influence the detection result.
- Sample Collection
- Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C
Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
- Sample Preparation
- Assay Procedure
- Prepare all reagents, samples and standards,
- Add 100μL standard or sample to each well. Incubate 2 hours at 37 °C,
- Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
- Aspirate and wash 3 times,
- Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
- Aspirate and wash 5 times,
- Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
- Add 50μL Stop Solution. Read at 450nm immediately.
- Prepare all reagents, samples and standards,
- Calculation of Results
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve on log-log graph paper, with LDB1 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
- Assay Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level LIM Domain Binding Protein 1 (LDB1) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level LIM Domain Binding Protein 1 (LDB1) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
- For Research Use only
- Precaution of Use
- The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
- Handling Advice
- The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.
- 4 °C
- Storage Comment
- For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
- Expiry Date
- 6 months