Mouse RAB35 (Ras-related protein Rab-35) ELISA Kit (MOES01818)
- SKU:
- MOES01818
Description
Mouse RAB35 (Ras-related protein Rab-35) 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 RAB35 . 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 RAB35 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 RAB35 , 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 RAB35 . The concentration of Mouse RAB35 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 | 78.13-5000 pg/mL |
| Sensitivity | 46.88 pg/mL |
| Sample Volume Required Per Well | 100uL |
| Sample Type | Serum, plasma and other biological fluids |
Specificity
This kit recognizes Mouse RAB35 in samples. No significant cross-reactivity or interference between Mouse RAB35 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 |
|---|---|---|---|
| 5000 | 2.425 2.469 | 2.447 | 2.387 |
| 2500 | 1.678 1.68 | 1.679 | 1.619 |
| 1250 | 0.981 0.963 | 0.972 | 0.912 |
| 625 | 0.506 0.542 | 0.524 | 0.464 |
| 312.5 | 0.266 0.24 | 0.253 | 0.193 |
| 156.25 | 0.175 0.165 | 0.17 | 0.11 |
| 78.13 | 0.107 0.125 | 0.116 | 0.056 |
| 0 | 0.059 0.061 | 0.06 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse RAB35 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse RAB35 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) | 248.86 | 458.66 | 2130.69 | 242.88 | 495.76 | 2084.59 |
| Standard deviation | 12.89 | 25.87 | 99.72 | 16.32 | 22.06 | 108.61 |
| C V (%) | 5.18 | 5.64 | 4.68 | 6.72 | 4.45 | 5.21 |
Recovery
The recovery of Mouse RAB35 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) | 88-102 | 94 |
| EDTA plasma (n=5) | 86-102 | 93 |
| Cell culture media (n=5) | 95-108 | 101 |
Linearity
Samples were spiked with high concentrations of Mouse RAB35 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 (%) | 93-107 | 95-112 | 97-108 |
| Average (%) | 100 | 102 | 103 | |
| 1:4 | Range (%) | 93-106 | 81-94 | 85-97 |
| Average (%) | 98 | 86 | 91 | |
| 1:8 | Range (%) | 86-99 | 80-93 | 85-96 |
| Average (%) | 93 | 87 | 91 | |
| 1:16 | Range (%) | 86-101 | 85-97 | 84-96 |
| Average (%) | 92 | 92 | 89 |
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 RAB35 (Ras-related protein Rab-35) ELISA Kit (MOES01818) 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.
Mouseà RAB35à (Ras-relatedà proteinà Rab-35)à ELISAà Kità à (MOES01818) Protein Information
| UniProt Protein Function: | RAB35: In the process of endocytosis, essential rate-limiting regulator of a fast recycling pathway back to the plasma membrane. During cytokinesis, required for the postfurrowing terminal steps, namely for intercellular bridge stability and abscission, possibly by controlling phosphatidylinositol 4,5-bis phosphate (PIP2) and SEPT2 localization at the intercellular bridge. Interacts with DENND1A and DENND1B in a nucleotide- dependent manner. The low binding to DENND1C is nucleotide- independent. All 3 DENND1 act as GEFs for RAB35 and thus activate the protein. Belongs to the small GTPase superfamily. Rab family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:G protein, monomeric, Rab; G protein, monomeric; G protein Chromosomal Location of Human Ortholog: 12q24.31 Cellular Component: Golgi membrane; endoplasmic reticulum membrane; intercellular bridge; mitochondrion; cell projection membrane; melanosome; plasma membrane; endosome membrane; coated pit; clathrin-coated endocytic vesicle Molecular Function:GTPase activity; protein binding; phosphatidylinositol-4,5-bisphosphate binding; GDP binding; GTP binding Biological Process: intracellular protein transport; antigen processing and presentation; ER to Golgi vesicle-mediated transport; protein localization; metabolic process; cytokinesis; plasma membrane to endosome transport; endosome transport; endocytic recycling; Rab protein signal transduction; neurite development |
| UniProt Code: | Q6PHN9 |
| NCBI GenInfo Identifier: | 262527293 |
| NCBI Gene ID: | 11021 |
| NCBI Accession: | NP_001161078.1 |
| UniProt Secondary Accession: | Q6PHN9,Q5U316, Q6PHN9, |
| UniProt Related Accession: | Q15286 |
| Molecular Weight: | 23025 |
| NCBI Full Name: | ras-related protein Rab-35 isoform 2 |
| NCBI Synonym Full Names: | RAB35, member RAS oncogene family |
| NCBI Official Symbol: | RAB35 |
| NCBI Official Synonym Symbols: | RAY; H-ray; RAB1C |
| NCBI Protein Information: | ras-related protein Rab-35 |
| UniProt Protein Name: | Ras-related protein Rab-35 |
| UniProt Synonym Protein Names: | GTP-binding protein RAY; Ras-related protein Rab-1C |
| Protein Family: | Ras-related protein |
| UniProt Gene Name: | RAB35 |
| UniProt Entry Name: | RAB35_HUMAN |
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