Mouse ACF (Apobec 1 Complementation Factor) ELISA Kit (MOES00719)
- SKU:
- MOES00719
Description
Mouse ACF (Apobec 1 Complementation Factor) ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse ACF . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse ACF and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse ACF, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse ACF. The concentration of Mouse ACF in samples can be calculated by comparing the OD of the samples to the standard curve.
| Assay type | Sandwich |
| Format | 96T |
| Assay time | 4.5h |
| Reactivity | Mouse |
| Detection Method | Colormetric |
| Detection Range | 31.25-2000 pg/mL |
| Sensitivity | 18.75 pg/mL |
| Sample Volume Required Per Well | 100uL |
| Sample Type | Serum, plasma and other biological fluids |
Specificity
This kit recognizes Mouse ACF in samples. No significant cross-reactivity or interference between Mouse ACF and analogues was observed.
Typical Data
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (pg/mL) | O.D | Average | Corrected |
|---|---|---|---|
| 2000 | 2.446 2.47 | 2.458 | 2.398 |
| 1000 | 1.582 1.618 | 1.6 | 1.54 |
| 500 | 0.913 0.899 | 0.906 | 0.846 |
| 250 | 0.444 0.456 | 0.45 | 0.39 |
| 125 | 0.24 0.222 | 0.231 | 0.171 |
| 62.5 | 0.175 0.149 | 0.162 | 0.102 |
| 31.25 | 0.11 0.114 | 0.112 | 0.052 |
| 0 | 0.052 0.068 | 0.06 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse ACF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse ACF were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 96.39 | 298.09 | 899.19 | 105.19 | 306.22 | 952.74 |
| Standard deviation | 5.99 | 14.31 | 35.70 | 6.86 | 15.10 | 35.16 |
| C V (%) | 6.21 | 4.80 | 3.97 | 6.52 | 4.93 | 3.69 |
Recovery
The recovery of Mouse ACF spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum (n=5) | 88-100 | 93 |
| EDTA plasma (n=5) | 86-101 | 93 |
| Cell culture media (n=5) | 95-106 | 101 |
Linearity
Samples were spiked with high concentrations of Mouse ACF and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 92-104 | 90-105 | 92-105 |
| Average (%) | 99 | 97 | 99 | |
| 1:4 | Range (%) | 90-105 | 87-98 | 84-97 |
| Average (%) | 98 | 93 | 91 | |
| 1:8 | Range (%) | 90-103 | 83-96 | 84-98 |
| Average (%) | 95 | 88 | 91 | |
| 1:16 | Range (%) | 88-100 | 80-93 | 87-102 |
| Average (%) | 95 | 86 | 93 |
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
|---|---|---|
| Micro ELISA Plate(Dismountable) | 8 wells X12 strips | -20'C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100X) | 1 vial, 120 uL | |
| Concentrated HRP Conjugate (100X) | 1 vial, 120 uL | -20'C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4'C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25X) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4'C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4'C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
Mouse ACF (Apobec 1 Complementation Factor) ELISA Kit (MOES00719) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Mouse ACF (Apobec 1 Complementation Factor) ELISA Kit (MOES00719) Protein Information
| UniProt Protein Function: | ACF: Essential component of the apolipoprotein B mRNA editing enzyme complex which is responsible for the postranscriptional editing of a CAA codon for Gln to a UAA codon for stop in APOB mRNA. Binds to APOB mRNA and is probably responsible for docking the catalytic subunit, APOBEC1, to the mRNA to allow it to deaminate its target cytosine. The complex also protects the edited APOB mRNA from nonsense-mediated decay. 6 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:RNA processing; RNA-binding; Hydrolase Chromosomal Location of Human Ortholog: 10q11.23 Cellular Component: nucleoplasm; endoplasmic reticulum; cytoplasm; apolipoprotein B mRNA editing enzyme complex Molecular Function:protein binding; single-stranded RNA binding; RNA binding; double-stranded RNA binding; nucleotide binding Biological Process: protein stabilization; cytidine to uridine editing; mRNA modification; gene expression; mRNA processing |
| NCBI Summary: | Mammalian apolipoprotein B mRNA undergoes site-specific C to U deamination, which is mediated by a multi-component enzyme complex containing a minimal core composed of APOBEC-1 and a complementation factor encoded by this gene. The gene product has three non-identical RNA recognition motifs and belongs to the hnRNP R family of RNA-binding proteins. It has been proposed that this complementation factor functions as an RNA-binding subunit and docks APOBEC-1 to deaminate the upstream cytidine. Studies suggest that the protein may also be involved in other RNA editing or RNA processing events. Several transcript variants encoding a few different isoforms have been found for this gene. [provided by RefSeq, Nov 2010] |
| UniProt Code: | Q5YD48 |
| NCBI GenInfo Identifier: | 219521544 |
| NCBI Gene ID: | 29974 |
| NCBI Accession: | AAI44197.1 |
| UniProt Secondary Accession: | Q5YD48,Q5YD48, |
| UniProt Related Accession: | Q9NQ94 |
| Molecular Weight: | 65,202 Da |
| NCBI Full Name: | A1CF protein |
| NCBI Synonym Full Names: | APOBEC1 complementation factor |
| NCBI Official Symbol: | A1CF |
| NCBI Official Synonym Symbols: | ACF; ASP; ACF64; ACF65; APOBEC1CF; RP11-564C4.2 |
| NCBI Protein Information: | APOBEC1 complementation factor; apo-B RNA editing protein; APOBEC-1 stimulating protein; apobec-1 complementation factor (ACF) (ASP) |
| UniProt Protein Name: | APOBEC1 complementation factor |
| UniProt Synonym Protein Names: | APOBEC1-stimulating protein |
| UniProt Gene Name: | A1CF |
| UniProt Entry Name: | A1CF_HUMAN |
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