Mouse Cell Signalling ELISA Kits 6

Mouse Runt-related transcription factor 2 (Runx2) ELISA Kit

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SKU:
MOEB2347
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q08775
ELISA Type:
Sandwich
Synonyms:
RUNX2, CBFA1, Acute myeloid leukemia 3 protein
Reactivity:
Mouse

Description

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Product Name:Mouse Runt-related transcription factor 2 (Runx2) ELISA Kit
Product Code:MOEB2347
Alias:Runt-related transcription factor 2, Acute myeloid leukemia 3 protein, Core-binding factor subunit alpha-1, CBF-alpha-1, Oncogene AML-3, Osteoblast-specific transcription factor 2, OSF-2, Polyomavirus enhancer-binding protein 2 alpha A subunit, PEA2-alpha A, PEBP2-alpha A, SL3-3 enhancer factor 1 alpha A subunit, SL3/AKV core-binding factor alpha A subunit, Runx2, Aml3, Cbfa1, Osf2, Pebp2a
Uniprot:Q08775
Reactivity:Mouse
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:AML3: Transcription factor involved in osteoblastic differentiation and skeletal morphogenesis. Essential for the maturation of osteoblasts and both intramembranous and endochondral ossification. CBF binds to the core site, 5'- PYGPYGGT-3', of a number of enhancers and promoters, including murine leukemia virus, polyomavirus enhancer, T-cell receptor enhancers, osteocalcin, osteopontin, bone sialoprotein, alpha 1(I) collagen, LCK, IL-3 and GM-CSF promoters. In osteoblasts, supports transcription activation: synergizes with SPEN/MINT to enhance FGFR2-mediated activation of the osteocalcin FGF-responsive element (OCFRE). Inhibits KAT6B-dependent transcriptional activation. Interaction with SATB2 results in enhanced DNA binding and transactivation by these transcription factors. Heterodimer of an alpha and a beta subunit. Interacts with HIVEP3 and HIPK3. The alpha subunit binds DNA as a monomer and through the Runt domain. DNA-binding is increased by heterodimerization. Interacts with XRCC6 (Ku70) and XRCC5 (Ku80). Interacts with KAT6A and KAT6B. Binds to cyclin B1 CCNB1. Interacts with DDX5. Specifically expressed in osteoblasts. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription factor; DNA-binding

Chromosomal Location of Human Ortholog: 6p21

Cellular Component: nucleoplasm; transcription factor complex; cytoplasm; nuclear chromatin

Molecular Function:protein domain specific binding; protein binding; bHLH transcription factor binding; chromatin binding; transcription factor activity; ATP binding

Biological Process: embryonic forelimb morphogenesis; transcription initiation from RNA polymerase II promoter; ossification; positive regulation of transcription, DNA-dependent; cell maturation; regulation of fibroblast growth factor receptor signaling pathway; chondrocyte development; embryonic cranial skeleton morphogenesis; stem cell differentiation; osteoblast development; odontogenesis of dentine-containing teeth; BMP signaling pathway; osteoblast differentiation; positive regulation of osteoblast differentiation; positive regulation of chondrocyte differentiation; negative regulation of smoothened signaling pathway; positive regulation of cell proliferation; gene expression; negative regulation of transcription, DNA-dependent; regulation of odontogenesis of dentine-containing teeth; osteoblast fate commitment; T cell differentiation; endochondral ossification

Disease: Metaphyseal Dysplasia With Maxillary Hypoplasia With Or Without Brachydactyly; Cleidocranial Dysplasia

NCBI Summary:This gene is a member of the RUNX family of transcription factors and encodes a nuclear protein with an Runt DNA-binding domain. This protein is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. The protein can bind DNA both as a monomer or, with more affinity, as a subunit of a heterodimeric complex. Two regions of potential trinucleotide repeat expansions are present in the N-terminal region of the encoded protein, and these and other mutations in this gene have been associated with the bone development disorder cleidocranial dysplasia (CCD). Transcript variants that encode different protein isoforms result from the use of alternate promoters as well as alternate splicing. [provided by RefSeq, Jul 2016]
UniProt Code:Q08775
NCBI GenInfo Identifier:17368460
NCBI Gene ID:860
NCBI Accession:Q13950.2
UniProt Secondary Accession:Q08775,Q08775, Q9Z2J9,
UniProt Related Accession:Q13950
Molecular Weight:
NCBI Full Name:Runt-related transcription factor 2
NCBI Synonym Full Names:RUNX family transcription factor 2
NCBI Official Symbol:RUNX2  
NCBI Official Synonym Symbols:CCD; AML3; CCD1; CLCD; OSF2; CBFA1; OSF-2; PEA2aA; PEBP2aA; CBF-alpha-1  
NCBI Protein Information:runt-related transcription factor 2
UniProt Protein Name:Runt-related transcription factor 2
UniProt Synonym Protein Names:Acute myeloid leukemia 3 protein; Core-binding factor subunit alpha-1; CBF-alpha-1; Oncogene AML-3; Osteoblast-specific transcription factor 2; OSF-2; Polyomavirus enhancer-binding protein 2 alpha A subunit; PEA2-alpha A; PEBP2-alpha A; SL3-3 enhancer factor 1 alpha A subunit; SL3/AKV core-binding factor alpha A subunit
Protein Family:Runt-related transcription factor
UniProt Gene Name:RUNX2  
UniProt Entry Name:RUNX2_HUMAN
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.
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