Epigenetics and Nuclear Signaling
LMX1B Colorimetric Cell-Based ELISA
- SKU:
- CBCAB00943
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Epigenetics and Nuclear Signaling
- Reactivity:
- Human
- Reactivity:
- Mouse
- Detection Method:
- Colorimetric
Description
| Product Name: | LMX1B Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00943 |
| ELISA Type: | Cell-Based |
| Target: | LMX1B |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The LMX1B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect LMX1B protein expression profile in cells. The kit can be used for measuring the relative amounts of LMX1B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on LMX1B.
Qualitative determination of LMX1B concentration is achieved by an indirect ELISA format. In essence, LMX1B is captured by LMX1B-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: 4010, UniProt ID: O60663, OMIM: 161200/602575, Unigene: Hs.129133 |
| Gene Symbol: | LMX1B |
| Sub Type: | None |
| UniProt Protein Function: | LMX1B: Essential for the specification of dorsal limb fate at both the zeugopodal and autopodal levels. Defects in LMX1B are the cause of nail-patella syndrome (NPS); also known as onychoosteodysplasia. NPS is a disease that cause abnormal skeletal patterning and renal dysplasia. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cell development/differentiation; DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 9q33.3 Cellular Component: nucleus Molecular Function:protein binding; zinc ion binding; sequence-specific DNA binding; transcription factor activity Biological Process: collagen fibril organization; transcription, DNA-dependent; in utero embryonic development; multicellular organismal development; neuron migration; cerebellum morphogenesis; limb morphogenesis; neuron differentiation; cell proliferation; organ growth; regulation of transcription, DNA-dependent; dorsal/ventral pattern formation; midbrain development; positive regulation of transcription from RNA polymerase II promoter; central nervous system neuron development Disease: Nail-patella Syndrome |
| NCBI Summary: | This gene encodes a member of LIM-homeodomain family of proteins containing two N-terminal zinc-binding LIM domains, 1 homeodomain, and a C-terminal glutamine-rich domain. It functions as a transcription factor, and is essential for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. Mutations in this gene are associated with nail-patella syndrome. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2010] |
| UniProt Code: | O60663 |
| NCBI GenInfo Identifier: | 485956425 |
| NCBI Gene ID: | 4010 |
| NCBI Accession: | O60663.3 |
| UniProt Secondary Accession: | O60663,O75463, Q5JU95, Q6ISC9, F8W7W6, |
| UniProt Related Accession: | O60663 |
| Molecular Weight: | 402 |
| NCBI Full Name: | LIM homeobox transcription factor 1-beta |
| NCBI Synonym Full Names: | LIM homeobox transcription factor 1, beta |
| NCBI Official Symbol: | LMX1B |
| NCBI Official Synonym Symbols: | NPS1; LMX1.2 |
| NCBI Protein Information: | LIM homeobox transcription factor 1-beta; LMX-1.2; LIM/homeobox protein 1.2; LIM/homeobox protein LMX1B |
| UniProt Protein Name: | LIM homeobox transcription factor 1-beta |
| UniProt Synonym Protein Names: | LIM/homeobox protein 1.2; LMX-1.2; LIM/homeobox protein LMX1B |
| Protein Family: | LIM/homeobox protein |
| UniProt Gene Name: | LMX1B |
| UniProt Entry Name: | LMX1B_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-LMX1B 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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