Description
hnRNP H Colorimetric Cell-Based ELISA
The HNRNP H Colorimetric Cell-Based ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of HNRNP H levels in various cell types and supernatants. This kit offers reliable and reproducible results, making it an invaluable tool for studying the role of HNRNP H in cellular processes.HNRNP H is a key RNA-binding protein involved in regulating gene expression, splicing, and mRNA metabolism. Dysregulation of HNRNP H has been linked to various diseases, including cancer, autoimmune disorders, and neurodegenerative conditions.
Understanding the function of HNRNP H can provide insights into disease mechanisms and potential therapeutic targets.With the HNRNP H Colorimetric Cell-Based ELISA Kit, researchers can accurately measure HNRNP H levels in cell samples, allowing for in-depth analysis of its biological functions and potential implications in disease pathogenesis. This kit is an essential tool for researchers studying RNA biology, gene regulation, and disease mechanisms.
| Product Name: | hnRNP H Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01044 |
| ELISA Type: | Cell-Based |
| Target: | hnRNP H |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The hnRNP H Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect hnRNP H protein expression profile in cells. The kit can be used for measuring the relative amounts of hnRNP H in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on hnRNP H.
Qualitative determination of hnRNP H concentration is achieved by an indirect ELISA format. In essence, hnRNP H is captured by hnRNP H-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: 3188, UniProt ID: P55795, OMIM: 300610, Unigene: Hs.632828 |
| Gene Symbol: | HNRNPH2 |
| Sub Type: | None |
| UniProt Protein Function: | hnRNP H2: This protein is a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes which provide the substrate for the processing events that pre-mRNAs undergo before becoming functional, translatable mRNAs in the cytoplasm. Binds poly(RG). |
| UniProt Protein Details: | Protein type:RNA-binding; RNA splicing Chromosomal Location of Human Ortholog: Xq22 Cellular Component: cytoplasm; membrane; nucleoplasm; nucleus; ribonucleoprotein complex Molecular Function:protein binding; RNA binding Biological Process: gene expression; nuclear mRNA splicing, via spliceosome |
| NCBI Summary: | This gene belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all of the hnRNPs are present in the nucleus some seem to shuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acid binding properties. The protein encoded by this gene has three repeats of quasi-RRM domains that binds to RNAs. It is very similar to the family member HNRPH1. This gene is thought to be involved in Fabray disease and X-linked agammaglobulinemia phenotype. Alternative splicing results in multiple transcript variants encoding the same protein. Read-through transcription between this locus and the ribosomal protein L36a gene has been observed. [provided by RefSeq, Jan 2011] |
| UniProt Code: | P55795 |
| NCBI GenInfo Identifier: | 2500576 |
| NCBI Gene ID: | 3188 |
| NCBI Accession: | P55795.1 |
| UniProt Secondary Accession: | P55795,Q9HHA7, A1L400, |
| UniProt Related Accession: | P55795 |
| Molecular Weight: | 49,264 Da |
| NCBI Full Name: | Heterogeneous nuclear ribonucleoprotein H2 |
| NCBI Synonym Full Names: | heterogeneous nuclear ribonucleoprotein H2 (H') |
| NCBI Official Symbol: | HNRNPH2Â Â |
| NCBI Official Synonym Symbols: | FTP3; HNRPH'; HNRPH2; hnRNPH'Â Â |
| NCBI Protein Information: | heterogeneous nuclear ribonucleoprotein H2 |
| UniProt Protein Name: | Heterogeneous nuclear ribonucleoprotein H2 |
| UniProt Synonym Protein Names: | FTP-3; Heterogeneous nuclear ribonucleoprotein H'; hnRNP H' |
| Protein Family: | Heterogeneous nuclear ribonucleoprotein |
| UniProt Gene Name: | HNRNPH2Â Â |
| UniProt Entry Name: | HNRH2_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-hnRNP H 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.)
