ALKBH5 Overexpression Lysate

Western Blot: ALKBH5 Overexpression Lysate (Adult Normal) [NBL1-07476] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for ALKBH5.

• Reactivity: Hu
• Applications: WB

ALKBH5 Overexpression Lysate Summary

• Description:

  ALKBH5 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC209110
  
  Accession#: NM_017758
  
  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: ALKBH5

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: 44.1 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 ALKBH5 Overexpression Lysate

• ABH5
• ABH5OFOXD
• alkB, alkylation repair homolog 5 (E. coli)
• ALKBH5
• Alkylated DNA repair protein alkB homolog 5
• EC 1.14.11.-
• EC 1.14.11.53
• FLJ20308
• OFOXD1
• oxoglutarate and iron-dependent oxygenase domain containing
• probable alpha-ketoglutarate-dependent dioxygenase ABH5

Background

ALKBH5, RNA demethylase ALKBH5, ALKB homolog 5 (44 kDa) is a dioxygenase enzyme which demethylates RNA in a reaction requiring oxygen, alpha-ketoglutarate, and iron. ALKBH5 and fat mass and obesity-associated protein (FTO) are involved in the demethylation of N6-methyladenosine or m6A mRNA, the most common post-translational modification in eukaryotic mRNA. m6A mRNA modifications are very dynamic; they are introduced into the mRNA by m6A methyltransferases (e.g., METTL3/14 and KIAA1429), removed by erasers such as FTO and ALKBH5, and decoded by m6A binding proteins (e.g., IGF2BP1 and YTHDF1/2/3). m6A mRNA modifications play roles in regulating mRNA splicing, processing, translation and stability. m6A mRNA modifications are also associated with pathological states including cancer (1, 2).

ALKBH5 has been implicated in various physiological and pathophysiological processes. In mice, ALKBH5 is highly expressed in the testes where loss of ALKBH5 expression leads to decreased spermatogenesis, abnormal spermatozoa and increased apoptosis (3). In glioblastoma, ALKBH5 overexpression promotes stemness and tumor progression (2). In breast cancer cells under hypoxic conditions, HIF-1 alpha and HIF-2 alpha induce the expression of ALKH5 and leads to increased demethylation and expression of Nanog thus promoting breast cancer stem cell properties (4).

References

1. Chen, X. Y., Zhang, J., & Zhu, J. S. (2019). The role of m6A RNA methylation in human cancer. Molecular Cancer. https://doi.org/10.1186/s12943-019-1033-z

2. Deng, X., Su, R., Weng, H., Huang, H., Li, Z., & Chen, J. (2018). RNA N 6 -methyladenosine modification in cancers: Current status and perspectives. Cell Research. https://doi.org/10.1038/s41422-018-0034-6

3. Meyer, K. D., & Jaffrey, S. R. (2014). The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/nrm3785

4. Zhang, C., Samanta, D., Lu, H., Bullen, J. W., Zhang, H., Chen, I., ... Semenza, G. L. (2016). Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA. Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.1602883113

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.

Bioinformatics

• Gene Symbol: ALKBH5