Signal Transduction
60S Ribosomal Protein L10 Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00508
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Signal Transduction
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | 60S Ribosomal Protein L10 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00508 |
| ELISA Type: | Cell-Based |
| Target: | 60S Ribosomal Protein L10 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The 60S Ribosomal Protein L10 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect 60S Ribosomal Protein L10 protein expression profile in cells. The kit can be used for measuring the relative amounts of 60S Ribosomal Protein L10 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on 60S Ribosomal Protein L10.
Qualitative determination of 60S Ribosomal Protein L10 concentration is achieved by an indirect ELISA format. In essence, 60S Ribosomal Protein L10 is captured by 60S Ribosomal Protein L10-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: 6134, UniProt ID: P27635, OMIM: 312173, Unigene: Hs.401929/Hs.534404/Hs.657751 |
| Gene Symbol: | RPL10 |
| Sub Type: | None |
| UniProt Protein Function: | RPL10: Defects in RPL10 are a cause of susceptibility to autism X-linked type 5 (AUTSX5). A complex multifactorial, pervasive developmental disorder characterized by impairments in reciprocal social interaction and communication, restricted and stereotyped patterns of interests and activities, and the presence of developmental abnormalities by 3 years of age. Most individuals with autism also manifest moderate mental retardation. RPL10 is involved in autism only in rare cases. Two hypomorphic variants affecting the translation process have been found in families with autism spectrum disorders, suggesting that aberrant translation may play a role in disease mechanisms. Belongs to the ribosomal protein L10e family. |
| UniProt Protein Details: | Protein type:Translation; Ribosomal Chromosomal Location of Human Ortholog: Xq28 Cellular Component: cytosol; endoplasmic reticulum; membrane Molecular Function:protein binding; structural constituent of ribosome Biological Process: mRNA catabolic process, nonsense-mediated decay; ribosomal large subunit assembly and maintenance; rRNA processing; SRP-dependent cotranslational protein targeting to membrane; translation; translational initiation; viral transcription Disease: Autism, Susceptibility To, X-linked 5 |
| UniProt Code: | P27635 |
| NCBI GenInfo Identifier: | 148887414 |
| NCBI Gene ID: | 6134 |
| NCBI Accession: | P27635.4 |
| UniProt Secondary Accession: | P27635,Q16470, Q2HXT7, Q53FH7, Q6FGN8, Q8TDA5, A3KQT0 D3DWW6, |
| Molecular Weight: | 24,604 Da |
| NCBI Full Name: | 60S ribosomal protein L10 |
| UniProt Protein Name: | 60S ribosomal protein L10 |
| UniProt Synonym Protein Names: | Laminin receptor homolog; Protein QM; Tumor suppressor QM |
| Protein Family: | 60S ribosomal protein |
| UniProt Gene Name: | RPL10 |
| UniProt Entry Name: | RL10_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-60S Ribosomal Protein L10 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.)
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