Mouse DNA damage-inducible transcript 3 protein (Ddit3) ELISA Kit
The Mouse DNA Damage-Inducible Transcript 3 Protein (DDIT3) ELISA Kit is a powerful tool for accurately measuring levels of DDIT3 in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and consistent results for a variety of research applications.DDIT3, also known as CHOP, is a key regulator in the cellular response to DNA damage and ER stress.
It is involved in apoptosis, cell cycle regulation, and cellular stress responses, making it a valuable target for studying diseases such as cancer, diabetes, and neurodegenerative disorders. The Mouse DDIT3 ELISA Kit provides researchers with a reliable method for investigating the role of DDIT3 in these diseases and potential therapeutic interventions.
Product Name:
Mouse DNA damage-inducible transcript 3 protein (Ddit3) ELISA Kit
SKU:
MOEB2357
Size:
96T
Target:
Mouse DNA damage-inducible transcript 3 protein (Ddit3)
Synonyms:
C/EBP zeta, C/EBP-homologous protein, C/EBP-homologous protein 10, CCAAT/enhancer-binding protein homologous protein, Growth arrest and DNA-damage-inducible protein GADD153, CHOP, CHOP-10, DDIT-3, Chop, Chop10, Gadd153
Assay Type:
Competitive
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
1.56-100ng/mL
Sensitivity:
0.66ng/mL
Intra CV:
6.9%
Inter CV:
10.4%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
95-105%
108-117%
89-99%
107-119%
EDTA Plasma(N=5)
91-101%
104-114%
97-109%
96-108%
Heparin Plasma(N=5)
85-97%
108-118%
102-111%
100-112%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
110
104-116
Plasma
112
106-118
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.
Uniprot:
P35639
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse DNA damage-inducible transcript 3 protein
Sub Unit:
Heterodimer. Interacts with TCF7L2/TCF4, EP300/P300, HDAC5 and HDAC6. Interacts with TRIB3 which blocks its association with EP300/P300. Interacts with FOXO3, CEBPB and ATF4 (By similarity). Interacts with HDAC1.
Research Area:
Cancer
Subcellular Location:
Cytoplasm Nucleus Present in the cytoplasm under non-stressed conditions and ER stress leads to its nuclear accumulation.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
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.Protein type: DNA-binding; Oncoprotein; Transcription factor; AutophagyCellular Component: cytoplasm; late endosome; nucleusMolecular Function: cAMP response element binding protein binding; DNA binding; leucine zipper domain binding; protein binding; protein heterodimerization activity; sequence-specific DNA binding; transcription corepressor activity; transcription factor activity; transcription factor bindingBiological Process: apoptosis; blood vessel maturation; cell cycle; cell cycle arrest; cell redox homeostasis; ER overload response; inhibition of CREB transcription factor; mRNA transcription from RNA polymerase II promoter; negative regulation of DNA binding; negative regulation of fat cell differentiation; negative regulation of myoblast differentiation; 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 apoptosis; 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, DNA-dependent; release of sequestered calcium ion into cytosol; response to starvation; response to unfolded protein; transcription, DNA-dependent; unfolded protein response; Wnt receptor signaling pathway
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_MOUSE
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
