Description
PGBD1 Colorimetric Cell-Based ELISA
The PGBD1 Colorimetric Cell-Based ELISA Kit is specifically designed for the rapid and accurate detection of PGBD1 levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a variety of research applications.PGBD1 is a protein that plays a crucial role in DNA transposition and genome evolution. It is involved in regulating gene expression and has been linked to various diseases such as cancer and genetic disorders.
Understanding the levels of PGBD1 in cells can provide valuable insights into disease mechanisms and potential therapeutic targets.With its easy-to-use protocol and quick results, the PGBD1 Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers studying cellular dynamics and gene regulation. Whether investigating basic biological processes or exploring disease mechanisms, this kit provides a reliable and efficient method for analyzing PGBD1 expression levels.
| Product Name: | PGBD1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01180 |
| ELISA Type: | Cell-Based |
| Target: | PGBD1 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The PGBD1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PGBD1 protein expression profile in cells. The kit can be used for measuring the relative amounts of PGBD1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PGBD1.
Qualitative determination of PGBD1 concentration is achieved by an indirect ELISA format. In essence, PGBD1 is captured by PGBD1-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: 84547, UniProt ID: Q96JS3, OMIM: 0, Unigene: Hs.144527 |
| Gene Symbol: | PGBD1 |
| Sub Type: | None |
| UniProt Protein Function: | PGBD1: |
| UniProt Protein Details: | Protein type:Unknown function Chromosomal Location of Human Ortholog: 6p22.1 Cellular Component: nucleus Molecular Function:protein binding; sequence-specific DNA binding; transcription factor activity |
| NCBI Summary: | The piggyBac family of proteins, found in diverse animals, are transposases related to the transposase of the canonical piggyBac transposon from the moth, Trichoplusia ni. This family also includes genes in several genomes, including human, that appear to have been derived from the piggyBac transposons. This gene belongs to the subfamily of piggyBac transposable element derived (PGBD) genes. The PGBD proteins appear to be novel, with no obvious relationship to other transposases, or other known protein families. This gene product is specifically expressed in the brain, however, its exact function is not known. Alternative splicing results in multiple transcript variants encoding the same protein.[provided by RefSeq, May 2010] |
| UniProt Code: | Q96JS3 |
| NCBI GenInfo Identifier: | 74751967 |
| NCBI Gene ID: | 84547 |
| NCBI Accession: | Q96JS3.1 |
| UniProt Secondary Accession: | Q96JS3,Q53F43, Q6NTF5, Q8WWS4, |
| UniProt Related Accession: | Q96JS3 |
| Molecular Weight: | 92,515 Da |
| NCBI Full Name: | PiggyBac transposable element-derived protein 1 |
| NCBI Synonym Full Names: | piggyBac transposable element derived 1 |
| NCBI Official Symbol: | PGBD1Â Â |
| NCBI Official Synonym Symbols: | SCAND4; HUCEP-4; dJ874C20.4Â Â |
| NCBI Protein Information: | piggyBac transposable element-derived protein 1 |
| UniProt Protein Name: | PiggyBac transposable element-derived protein 1 |
| UniProt Synonym Protein Names: | Cerebral protein 4 |
| Protein Family: | PiggyBac transposable element-derived protein |
| UniProt Gene Name: | PGBD1Â Â |
| UniProt Entry Name: | PGBD1_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-PGBD1 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.)
