Immunology
GANP Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00669
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Immunology
- Reactivity:
- Human
- Reactivity:
- Mouse
- Detection Method:
- Colorimetric
Description
system_update_altDatasheet
| Product Name: | GANP Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00669 |
| ELISA Type: | Cell-Based |
| Target: | GANP |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The GANP Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GANP protein expression profile in cells. The kit can be used for measuring the relative amounts of GANP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GANP.
Qualitative determination of GANP concentration is achieved by an indirect ELISA format. In essence, GANP is captured by GANP-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: 8888, UniProt ID: O60318, OMIM: 603294, Unigene: Hs.389037 |
| Gene Symbol: | MCM3AP |
| Sub Type: | None |
| UniProt Protein Function: | GANP: is a member of the SAC3 family of nuclear export factors. In combination with ENY2, PCID2 and centrins, forms the TREX-2 complex that links transcription/ processing with nuclear mRNA export. GANP, required to recruit ENY2 to nuclear pore complexes (NPCs), requires both its CID and MCM3AP domains, together with nucleoporin Nup153, for recruitment to NPCs. The MCM3AP domain, corresponding to the carboxy-terminal 721 amino acids of GANP, shows homology with the Gcn5-related N acetyltransferase superfamily. The protein MCM3AP is encoded entirely within the 3¿ region of the GANP gene. MCM3AP transcription begins within intron 16 of GANP immediately upstream of the first MCM3AP exon, where NF-IL6, AP-1, NF-¿B, and p53 consensus transcription factor binding sites are located. In contrast, the GANP promoter region contains consensus transcription factor binding sites for E2F, E-box, BSAP, and PU.1. MCM3AP interacts with MCM3, which it can acetylate weakly in vivo. GANP is required for efficient mRNA nuclear export: GANP depletion results in the nuclear accumulation of poly(A)+RNA. It interacts via its N-terminal FG repeat domain with the mRNA export factor NXF1. GANP plays a role in the immune response: antibodies from mice deficient for GANP have reduced affinities against T-cell dependent antigens and a lower frequency of variable region somatic mutation. GANP expression is markedly decreased in malignant glioma (MG) with a poor prognosis, consistently lower than that observed in anaplastic astrocytomas. Significant decreases in GANP expression are associated with hyperploidy and chromosomal instability |
| UniProt Protein Details: | Protein type:Acetyltransferase; Karyopherin Chromosomal Location of Human Ortholog: 21q22.3 Cellular Component: nucleoplasm; nuclear membrane; cytoplasm; nuclear pore; nucleus; cytosol Molecular Function:DNA binding; transferase activity, transferring acyl groups Biological Process: mRNA transport; protein import into nucleus; immune system process; DNA replication |
| NCBI Summary: | The minichromosome maintenance protein 3 (MCM3) is one of the MCM proteins essential for the initiation of DNA replication. The protein encoded by this gene is a MCM3 binding protein. It was reported to have phosphorylation-dependent DNA-primase activity, which was up-regulated in antigen immunization induced germinal center. This protein was demonstrated to be an acetyltransferase that acetylates MCM3 and plays a role in DNA replication. The mutagenesis of a nuclear localization signal of MCM3 affects the binding of this protein with MCM3, suggesting that this protein may also facilitate MCM3 nuclear localization. This gene is expressed in the brain or in neuronal tissue. An allelic variant encoding amino acid Lys at 915, instead of conserved Glu, has been identified in patients with mild intellectual disability. [provided by RefSeq, Jan 2014] |
| UniProt Code: | O60318 |
| NCBI GenInfo Identifier: | 8134564 |
| NCBI Gene ID: | 8888 |
| NCBI Accession: | O60318.2 |
| UniProt Secondary Accession: | O60318,Q2M3C1, Q6PJP6, Q9BSY5, Q9UMT4, C9JL56, |
| UniProt Related Accession: | O60318 |
| Molecular Weight: | 80,292 Da |
| NCBI Full Name: | Germinal-center associated nuclear protein |
| NCBI Synonym Full Names: | minichromosome maintenance complex component 3 associated protein |
| NCBI Official Symbol: | MCM3AP |
| NCBI Official Synonym Symbols: | GANP; SAC3; MAP80 |
| NCBI Protein Information: | germinal-center associated nuclear protein; 80 kDa MCM3-associated protein; MCM3 acetylating protein; MCM3 acetyltransferase; MCM3 import protein; MCM3 minichromosome maintenance deficient 3 associated protein; germinal center-associated nuclear protein; germinal-centre associated nuclear protein |
| UniProt Protein Name: | Germinal-center associated nuclear protein |
| UniProt Synonym Protein Names: | 80 kDa MCM3-associated protein; MCM3 acetylating protein (EC:2.3.1.-); MCM3AP; MCM3 acetyltransferase |
| Protein Family: | Putative arabinogalactan oligomer transport system permease protein |
| UniProt Gene Name: | MCM3AP |
| UniProt Entry Name: | GANP_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-GANP 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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