Description
Human GADD153/DDIT3 ELISA Kit
The Human GADD153 (DDIT3) ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of GADD153 levels in human serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results, making it ideal for a variety of research applications.GADD153, also known as DDIT3, is a stress-responsive transcription factor involved in regulating cellular responses to various stress stimuli, including endoplasmic reticulum stress.
Dysregulation of GADD153 has been implicated in a number of diseases, including cancer, diabetes, and neurodegenerative disorders, making it a valuable biomarker for studying these conditions and potential therapeutic targets.Overall, the Human GADD153 (DDIT3) ELISA Kit is a valuable tool for researchers studying stress response pathways and the role of GADD153 in various diseases and conditions.
| Product Name: | Human GADD153/DDIT3 ELISA Kit |
| Product Code: | HUFI02084 |
| Size: | 96 Assays |
| Alias: | DDIT3, CEBP zeta, CHOP, CHOP10, C, EBP-homologous protein, C, EBP-homologous protein 10, CCAAT, enhancer-binding protein homologous protein, CHOP-10, CHOP10 CEBPZ, CHOPC, EBP zeta, DDIT-3, DNA damage-inducible transcript 3 protein, DNA-damage-inducible transcript 3, GADD153, Growth arrest and DNA damage-inducible protein GADD153 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human DDIT3 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 18.75pg/ml |
| Range: | 31.25-2000pg/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human DDIT3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human DDIT3 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human DDIT3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
| Uniprot | P35638 |
| UniProt Protein Function: | CHOP: a transcriptional-regulatory protein of the bZIP family. Inhibits the DNA-binding activity of C/EBP and LAP by forming heterodimers that cannot bind DNA. May play an important role in melanoma progression. CK2-mediated phosphorylation inhibits its transcriptional activity. Up-regulates IL-6 transcription by trapping negative regulating NF-IL6 isoform. |
| UniProt Protein Details: | Protein type:Autophagy; Oncoprotein; Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 12q13.1-q13.2 Cellular Component: cytosol; nucleoplasm; nucleus Molecular Function:cAMP response element binding protein binding; DNA binding; leucine zipper domain binding; protein binding; protein heterodimerization activity; protein homodimerization activity; transcription corepressor activity; transcription factor activity; transcription factor binding Biological Process: cell cycle arrest; cell redox homeostasis; inhibition of CREB transcription factor; mRNA transcription from RNA polymerase II promoter; negative regulation of protein kinase B signaling cascade; negative regulation of transcription factor activity; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of interleukin-8 production; positive regulation of neuron apoptosis; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; proteasomal ubiquitin-dependent protein catabolic process; regulation of transcription in response to stress; regulation of transcription, DNA-dependent; response to DNA damage stimulus; response to unfolded protein Disease: Myxoid Liposarcoma |
| NCBI Summary: | This gene encodes a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. The protein functions as a dominant-negative inhibitor by forming heterodimers with other C/EBP members, such as C/EBP and LAP (liver activator protein), and preventing their DNA binding activity. The protein is implicated in adipogenesis and erythropoiesis, is activated by endoplasmic reticulum stress, and promotes apoptosis. Fusion of this gene and FUS on chromosome 16 or EWSR1 on chromosome 22 induced by translocation generates chimeric proteins in myxoid liposarcomas or Ewing sarcoma. Multiple alternatively spliced transcript variants encoding two isoforms with different length have been identified. [provided by RefSeq, Aug 2010] |
| UniProt Code: | P35638 |
| NCBI GenInfo Identifier: | 544022 |
| NCBI Gene ID: | 1649 |
| NCBI Accession: | P35638.1 |
| UniProt Secondary Accession: | P35638,F8VS99, |
| UniProt Related Accession: | P35638 |
| Molecular Weight: | 21,700 Da |
| NCBI Full Name: | DNA damage-inducible transcript 3 protein |
| NCBI Synonym Full Names: | DNA damage inducible transcript 3 |
| NCBI Official Symbol: | DDIT3  |
| NCBI Official Synonym Symbols: | CHOP; CEBPZ; CHOP10; CHOP-10; GADD153  |
| NCBI Protein Information: | DNA damage-inducible transcript 3 protein |
| UniProt Protein Name: | DNA damage-inducible transcript 3 protein |
| UniProt Synonym Protein Names: | C/EBP zeta; C/EBP-homologous protein; CHOP; C/EBP-homologous protein 10; CHOP-10; CCAAT/enhancer-binding protein homologous protein; Growth arrest and DNA damage-inducible protein GADD153 |
| Protein Family: | DNA damage-inducible transcript 3 protein |
| UniProt Gene Name: | DDIT3  |
| UniProt Entry Name: | DDIT3_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
