Description
APLP2 Colorimetric Cell-Based ELISA Kit
The APLP2 Colorimetric Cell-Based ELISA Kit is a powerful tool for scientists and researchers looking to accurately measure the levels of Amyloid Precursor-Like Protein 2 (APLP2) in cell lysates and tissue samples. This kit offers high sensitivity and specificity, providing reliable and reproducible results for a wide range of experimental applications.APLP2 is a key protein involved in neuronal development and function, with implications in neurodegenerative diseases such as Alzheimer's.
By quantifying APLP2 levels, researchers can gain valuable insights into disease mechanisms and potentially identify new therapeutic targets.The APLP2 Colorimetric Cell-Based ELISA Kit is user-friendly and easy to use, making it a valuable addition to any lab's research toolkit. With its ability to detect APLP2 in a variety of sample types, this kit is a versatile and essential resource for studying the role of APLP2 in neurobiology and disease pathology.
| Product Name: | APLP2 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00273 |
| ELISA Type: | Cell-Based |
| Target: | APLP2 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The APLP2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect APLP2 protein expression profile in cells. The kit can be used for measuring the relative amounts of APLP2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on APLP2.
Qualitative determination of APLP2 concentration is achieved by an indirect ELISA format. In essence, APLP2 is captured by APLP2-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 334, UniProt ID: Q06481, OMIM: 104776, Unigene: Hs.370247 |
| Gene Symbol: | APLP2 |
| Sub Type: | None |
| UniProt Protein Function: | APLP2: May play a role in the regulation of hemostasis. The soluble form may have inhibitory properties towards coagulation factors. May interact with cellular G-protein signaling pathways. May bind to the DNA 5'-GTCACATG-3'(CDEI box). Inhibits trypsin, chymotrypsin, plasmin, factor XIA and plasma and glandular kallikrein. Modulates the Cu/Zn nitric oxide-catalyzed autodegradation of GPC1 heparan sulfate side chains in fibroblasts. Belongs to the APP family. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Receptor, misc.; Cell surface; DNA-binding; Membrane protein, integral Chromosomal Location of Human Ortholog: 11q24 Cellular Component: membrane; nucleus; plasma membrane; platelet alpha granule membrane Molecular Function:identical protein binding; protein binding Biological Process: platelet degranulation |
| NCBI Summary: | This gene encodes amyloid precursor- like protein 2 (APLP2), which is a member of the APP (amyloid precursor protein) family including APP, APLP1 and APLP2. This protein is ubiquitously expressed. It contains heparin-, copper- and zinc- binding domains at the N-terminus, BPTI/Kunitz inhibitor and E2 domains in the middle region, and transmembrane and intracellular domains at the C-terminus. This protein interacts with major histocompatibility complex (MHC) class I molecules. The synergy of this protein and the APP is required to mediate neuromuscular transmission, spatial learning and synaptic plasticity. This protein has been implicated in the pathogenesis of Alzheimer's disease. Multiple alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Aug 2011] |
| UniProt Code: | Q06481 |
| NCBI GenInfo Identifier: | 1703344 |
| NCBI Gene ID: | 334 |
| NCBI Accession: | Q06481.2 |
| UniProt Secondary Accession: | Q06481,Q13861, Q14594, Q14662, Q71U10, Q7M4L3, Q9BT36 B3KXX9, H7BXI4, |
| UniProt Related Accession: | Q06481 |
| Molecular Weight: | 87,215 Da |
| NCBI Full Name: | Amyloid-like protein 2 |
| NCBI Synonym Full Names: | amyloid beta precursor like protein 2 |
| NCBI Official Symbol: | APLP2 |
| NCBI Official Synonym Symbols: | APPH; APPL2; CDEBP; APLP-2 |
| NCBI Protein Information: | amyloid-like protein 2 |
| UniProt Protein Name: | Amyloid-like protein 2 |
| UniProt Synonym Protein Names: | APPH; Amyloid protein homolog; CDEI box-binding protein; CDEBP |
| Protein Family: | Amyloid-like protein |
| UniProt Gene Name: | APLP2 |
| UniProt Entry Name: | APLP2_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-APLP2 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
