ATP-Citrate Lyase (Phospho-Ser454) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01338
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Research Area:
- Metabolism
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | ATP-Citrate Lyase (Phospho-Ser454) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01338 |
| ELISA Type: | Cell-Based |
| Target: | ATP-Citrate Lyase (Phospho-Ser454) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The ATP-Citrate Lyase (Phospho-Ser454) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ATP-Citrate Lyase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ATP-Citrate Lyase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ATP-Citrate Lyase phosphorylation.
Qualitative determination of ATP-Citrate Lyase (Phospho-Ser454) concentration is achieved by an indirect ELISA format. In essence, ATP-Citrate Lyase (Phospho-Ser454) is captured by ATP-Citrate Lyase (Phospho-Ser454)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 47, UniProt ID: P53396, OMIM: 108728, Unigene: Hs.387567 |
| Gene Symbol: | ACLY |
| Sub Type: | Phospho |
| UniProt Protein Function: | ACLY: ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Has a central role in de novo lipid synthesis. In nervous tissue it may be involved in the biosynthesis of acetylcholine. Homotetramer. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:EC 2.3.3.8; Transferase; Lyase; Carbohydrate Metabolism - citrate (TCA) cycle Chromosomal Location of Human Ortholog: 17q21.2 Cellular Component: cytoplasm; cytosol; membrane; mitochondrion; nucleoplasm; plasma membrane Molecular Function:ATP binding; ATP citrate synthase activity; cofactor binding; metal ion binding; protein binding Biological Process: acetyl-CoA biosynthetic process; cellular lipid metabolic process; cholesterol biosynthetic process; citrate metabolic process; energy reserve metabolic process; fatty acid biosynthetic process; lipid biosynthetic process; oxaloacetate metabolic process; positive regulation of cellular metabolic process; triacylglycerol biosynthetic process |
| NCBI Summary: | ATP citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. The enzyme is a tetramer (relative molecular weight approximately 440,000) of apparently identical subunits. It catalyzes the formation of acetyl-CoA and oxaloacetate from citrate and CoA with a concomitant hydrolysis of ATP to ADP and phosphate. The product, acetyl-CoA, serves several important biosynthetic pathways, including lipogenesis and cholesterogenesis. In nervous tissue, ATP citrate-lyase may be involved in the biosynthesis of acetylcholine. Multiple transcript variants encoding distinct isoforms have been identified for this gene. [provided by RefSeq, Dec 2014] |
| UniProt Code: | P53396 |
| NCBI GenInfo Identifier: | 116241237 |
| NCBI Gene ID: | 47 |
| NCBI Accession: | P53396.3 |
| UniProt Secondary Accession: | P53396,Q13037, Q9BRL0, B4DIM0, B4E3P0, |
| UniProt Related Accession: | P53396 |
| Molecular Weight: | 91,099 Da |
| NCBI Full Name: | ATP-citrate synthase |
| NCBI Synonym Full Names: | ATP citrate lyase |
| NCBI Official Symbol: | ACLY |
| NCBI Official Synonym Symbols: | ACL; ATPCL; CLATP |
| NCBI Protein Information: | ATP-citrate synthase |
| UniProt Protein Name: | ATP-citrate synthase |
| UniProt Synonym Protein Names: | ATP-citrate (pro-S-)-lyase; ACL; Citrate cleavage enzyme |
| Protein Family: | ATP-citrate synthase |
| UniProt Gene Name: | ACLY |
| UniProt Entry Name: | ACLY_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-ATP-Citrate Lyase (Phospho-Ser454) Antibody, Anti-ATP-Citrate Lyase 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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