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Rat GADD153 / DDIT3 ELISA Kit

SKU:
RTFI00532
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q62857
Sensitivity:
0.375ng/ml
Range:
0.625-40ng/ml
ELISA Type:
Sandwich
Synonyms:
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, GADD153, Growth arrest and DNA damage-inducible protein GADD1
Reactivity:
Rat
Research Area:
Cell Death
€649
Frequently bought together:

Description

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Product Name:Rat Ddit3 (DNA damage-inducible transcript 3 protein) ELISA Kit
Product Code:RTFI00532
Size:96 Assays
Target:Rat Ddit3
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, GADD153, Growth arrest and DNA damage-inducible protein GADD153
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:0.375ng/ml
Range:0.625-40ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat Ddit3 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Ddit3 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)92-10597
EDTA plasma(n=5)85-10595
UFH plasma(n=5)91-10096
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Ddit3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-102%85-103%85-103%
EDTA plasma(n=5)82-101%84-101%87-100%
UFH plasma(n=5)88-98%81-97%83-96%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:Q62857
UniProt Protein Function:Multifunctional transcription factor in ER stress response. Plays an essential role in the response to a wide variety of cell stresses and induces cell cycle arrest and apoptosis in response to ER stress. Plays a dual role both as an inhibitor of CCAAT/enhancer-binding protein (C/EBP) function and as an activator of other genes. Acts as a dominant-negative regulator of C/EBP-induced transcription: dimerizes with members of the C/EBP family, impairs their association with C/EBP binding sites in the promoter regions, and inhibits the expression of C/EBP regulated genes. Positively regulates the transcription of TRIB3, IL6, IL8, IL23, TNFRSF10B/DR5, PPP1R15A/GADD34, BBC3/PUMA, BCL2L11/BIM and ERO1L. Negatively regulates; expression of BCL2 and MYOD1, ATF4-dependent transcriptional activation of asparagine synthetase (ASNS), CEBPA-dependent transcriptional activation of hepcidin (HAMP) and CEBPB-mediated expression of peroxisome proliferator-activated receptor gamma (PPARG). Inhibits the canonical Wnt signaling pathway by binding to TCF7L2/TCF4, impairing its DNA-binding properties and repressing its transcriptional activity. Plays a regulatory role in the inflammatory response through the induction of caspase-11 (CASP4/CASP11) which induces the activation of caspase-1 (CASP1) and both these caspases increase the activation of pro-IL1B to mature IL1B which is involved in the inflammatory response. Acts as a major regulator of postnatal neovascularization through regulation of endothelial nitric oxide synthase (NOS3)-related signaling.
NCBI Summary:plays a role in the ER stress response [RGD, Feb 2006]
UniProt Code:Q62857
NCBI GenInfo Identifier:81890307
NCBI Gene ID:29467
NCBI Accession:Q62857.1
UniProt Related Accession:Q62857
Molecular Weight:19,013 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:RM4; CHOP; 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  
StepProcedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.

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