Description
Tubulin beta Colorimetric Cell-Based ELISA Kit
The Tubulin Beta Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the quantitative detection of Tubulin Beta levels in cell lysates and cell culture supernatants. This kit offers high sensitivity and specificity for accurate and reproducible results, making it ideal for a variety of research applications.Tubulin Beta is a key component of microtubules, essential for cell division, intracellular transport, and cell structure. Dysregulation of Tubulin Beta has been implicated in various diseases, including cancer, neurodegenerative disorders, and developmental abnormalities.
By measuring Tubulin Beta levels, researchers can better understand the role of this protein in disease pathogenesis and potentially identify new therapeutic targets.With the Tubulin Beta Colorimetric Cell-Based ELISA Kit, researchers can delve deeper into the intricate mechanisms of cellular function and disease progression, paving the way for innovative discoveries and advancements in the field of biomedical research.
| Product Name: | Tubulin beta Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00090 |
| ELISA Type: | Cell-Based |
| Target: | Tubulin beta |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Tubulin beta Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Tubulin beta protein expression profile in cells. The kit can be used for measuring the relative amounts of Tubulin beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Tubulin beta.
Qualitative determination of Tubulin beta concentration is achieved by an indirect ELISA format. In essence, Tubulin beta is captured by Tubulin beta-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: 10381, UniProt ID: Q13509, OMIM: 602661, Unigene: Hs.511743 |
| Gene Symbol: | TUBB3 |
| Sub Type: | None |
| UniProt Protein Function: | TUBB3: Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha-chain. TUBB3 plays a critical role in proper axon guidance and mantainance. Dimer of alpha and beta chains. Expression is primarily restricted to central and peripheral nervous system. Greatly increased expression in most cancerous tissues. Belongs to the tubulin family. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Cytoskeletal Chromosomal Location of Human Ortholog: 16q24.3 Cellular Component: axon; cell soma; cytoplasm; dendrite; microtubule; nucleus Molecular Function:GTP binding; GTPase activity; peptide binding; protein binding; structural constituent of cytoskeleton Biological Process: 'de novo' posttranslational protein folding; axon guidance; cellular protein metabolic process; microtubule-based process; mitosis; protein folding Disease: Cortical Dysplasia, Complex, With Other Brain Malformations 1; Fibrosis Of Extraocular Muscles, Congenital, 3a, With Or Without Extraocular Involvement |
| NCBI Summary: | This gene encodes a class III member of the beta tubulin protein family. Beta tubulins are one of two core protein families (alpha and beta tubulins) that heterodimerize and assemble to form microtubules. This protein is primarily expressed in neurons and may be involved in neurogenesis and axon guidance and maintenance. Mutations in this gene are the cause of congenital fibrosis of the extraocular muscles type 3. Alternate splicing results in multiple transcript variants. A pseudogene of this gene is found on chromosome 6. [provided by RefSeq, Oct 2010] |
| UniProt Code: | Q13509 |
| NCBI GenInfo Identifier: | 20455526 |
| NCBI Gene ID: | 10381 |
| NCBI Accession: | Q13509.2 |
| UniProt Secondary Accession: | Q13509,Q9BTZ0, Q9BW10, A8K854, |
| UniProt Related Accession: | Q13509 |
| Molecular Weight: | 42,433 Da |
| NCBI Full Name: | Tubulin beta-3 chain |
| NCBI Synonym Full Names: | tubulin beta 3 class III |
| NCBI Official Symbol: | TUBB3 |
| NCBI Official Synonym Symbols: | CDCBM; FEOM3; TUBB4; CDCBM1; CFEOM3; beta-4; CFEOM3A |
| NCBI Protein Information: | tubulin beta-3 chain |
| UniProt Protein Name: | Tubulin beta-3 chain |
| UniProt Synonym Protein Names: | Tubulin beta-4 chain; Tubulin beta-III |
| Protein Family: | Tubulin |
| UniProt Gene Name: | TUBB3 |
| UniProt Entry Name: | TBB3_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-Tubulin beta 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.)
