RUNX2/CBFA1 Overexpression Lysate

Western Blot: RUNX2 Overexpression Lysate (Adult Normal) [NBL1-15628] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for RUNX2.

• Reactivity: Hu
• Applications: WB

RUNX2/CBFA1 Overexpression Lysate Summary

• Description:

  RUNX2/CBFA1 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC213097
  
  Accession#: NM_004348
  
  Protein Tag: C-MYC/DDK
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.
• Gene: RUNX2

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.
  
  Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.
• Theoretical MW: 54.9 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Alternate Names for RUNX2/CBFA1 Overexpression Lysate

• Acute myeloid leukemia 3 protein
• CBFA1
• CBF-alpha-1
• CCD1
• CCDAML3
• CLCD
• Core-binding factor subunit alpha-1
• core-binding factor, runt domain, alpha subunit 1
• MGC120023
• ML3
• oncogene AML-3
• OSF2
• OSF-2
• osteoblast-specific transcription factor 2
• PEA2aA
• PEA2-alpha A
• PEBP2A
• PEBP2aA
• PEBP2-alpha A
• polyomavirus enhancer-binding protein 2 alpha A subunit
• runt domain, alpha subunit 1
• runt related transcription factor 2
• runt-related transcription factor 2
• RUNX2
• SL3/AKV core-binding factor alpha A subunit
• SL3-3 enhancer factor 1 alpha A subunit

Background

Runt-related transcription factor 2 (RUNX2), also known as CBFA1, AML-3, PEBP-2alphaA, and OSF-2, is a transcription factor that places a critical role in osteoblast differentiation and bone development (1-3). RUNX2 is a DNA-binding protein that belongs to the RUNX family which share a common runt domain (3). RUNX2 has two main isoforms which vary based on the two promoter regions (3). The main canonical isoform (P1) has MASN/DS at its N-terminus while the other (P2) isoform includes a MRIPV pentapeptide at its N-terminus (3). The RUNX2 P1 isoform has a theoretical molecular weight of 56 kDa and is synthesized as a 521 amino acid (aa) protein containing multiple domains. Specifically, RUNX2 contains transactivation domains (AD1, 2 and 3), a glutamine/alanine (Q/A)-rich domain, a runt homology domain (RHD), a nuclear localization signal (NLS), a proline/serine/threonine (PST)-rich domain, a nuclear matrix targeting signal (NMTS), a repression domain (RD), and a VWRPY region (3). RUNX2 is a heterodimer of an alpha and beta subunit where the alpha subunit binds DNA through the runt domain and the binding affinity is increased through heterodimerization (4).

Functionally, RUNX2 promotes the expression of osteoblast-specific genes vital for the osteoblast differentiation and proliferation process including type I collagen, osteocalcin (OCN), and alkaline phosphatase (APC) (1, 3). Further evidence for the role of RUNX2 is highlighted by a study of Runx2-/-mice which completely lack osteoblasts (4). Additionally, RUNX2 is also required for chondrocyte maturation, which are the cells responsible for cartilage formation (1, 3, 5). Given the role of RUNX2 in bone and cartilage maturation and formation, it is clear that defects or mutations in RUNX2 cause various bone and bone-related diseases (3, 6, 7). For instance, cleidocranial dysplasia (CCD), which presents with delayed cranial suture closure phenotypes, hypoplastic clavicles, extra teeth, and short stature, is caused by haploinsufficiency in RUNX2 (2, 3, 6). Furthermore, metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is a bone dysplasia disorder with a phenotype of abnormalities in the long bones, an underdeveloped jawbone, and short fingers that is caused by a duplication in RUNX2 (6). Finally, RUNX2 has been shown to be upregulated in mouse models of the joint disorder osteoarthritis (OA) and may be a potential molecular target for disease treatment (7).

Alternative names for RUNX2 include Acute myeloid leukemia 3 protein CBFA1, CBF-alpha-1, CCD1, CCDAML3, CLCD, Core-binding factor subunit alpha-1, MGC120023, ML3, oncogene AML-3, OSF2, osteoblast-specific transcription factor 2, PEA2aA, PEA2-alpha A, PEBP2A, polyomavirus enhancer-binding protein 2 alpha A subunit, runt related transcription factor 2, SL3/AKV core-binding factor alpha A subunit, and SL3-3 enhancer factor 1 alpha A subunit.

References

1. Ferreira, L. B., Gimba, E., Vinagre, J., Sobrinho-Simoes, M., & Soares, P. (2020). Molecular Aspects of Thyroid Calcification. International journal of molecular sciences. https://doi.org/10.3390/ijms21207718

2. Kim, W. J., Shin, H. L., Kim, B. S., Kim, H. J., & Ryoo, H. M. (2020). RUNX2-modifying enzymes: therapeutic targets for bone diseases. Experimental & molecular medicine. https://doi.org/10.1038/s12276-020-0471-4

3. Vimalraj, S., Arumugam, B., Miranda, P. J., & Selvamurugan, N. (2015). Runx2: Structure, function, and phosphorylation in osteoblast differentiation. International journal of biological macromolecules. https://doi.org/10.1016/j.ijbiomac.2015.04.008

4. Uniprot (Q13950)

5. Komori T. (2017). Roles of Runx2 in Skeletal Development. Advances in experimental medicine and biology. https://doi.org/10.1007/978-981-10-3233-2_6

6. Moffatt, P., Ben Amor, M., Glorieux, F. H., Roschger, P., Klaushofer, K., Schwartzentruber, J. A., Paterson, A. D., Hu, P., Marshall, C., FORGE Canada Consortium, Fahiminiya, S., Majewski, J., Beaulieu, C. L., Boycott, K. M., & Rauch, F. (2013). Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly is caused by a duplication in RUNX2. American journal of human genetics. https://doi.org/10.1016/j.ajhg.2012.12.001

7. Chen, D., Kim, D. J., Shen, J., Zou, Z., & O'Keefe, R. J. (2019). Runx2 plays a central role in Osteoarthritis development. Journal of orthopaedic translation. https://doi.org/10.1016/j.jot.2019.11.008

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

FAQs for RUNX2/CBFA1 Lysate (NBL1-15628). (Showing 1 - 1 of 1 FAQ).

1. We would like an anti-RUNX2 for IHC-P which share cross reactivity with Rat, but not with Human.
   • We don't have any data for our RUNX2 antibodies that confirms they will NOT detect the human protein. When we can confirm that an antibody will not react with a certain species, we display a (-) sign on the datasheet. Otherwise, if the species is not listed it means that it has not been tested.

Bioinformatics

• Gene Symbol: RUNX2