Revision 5

#49015Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IF-IC

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

45-48

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q6P6C2

Entrez-Gene Id:

54890

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:3200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

ALKBH5 (E3F6E) Rabbit mAb recognizes endogenous levels of total ALKBH5 protein. This antibody is predicted to detect only isoforms 2 and 3 of ALKBH5. Reactivity by immunofluorescence is human only.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ALKBH5 protein.

Background

N6-methyladenosine (m6A) is an abundant and reversible RNA modification that plays an important role in mRNA splicing, processing, and stability. Demethylation of m6A is carried out by two enzymes, fat mass and obesity-related protein (FTO) and AlkB homolog 5 (ALKBH5). ALKBH5 belongs to the AlkB subfamily of Fe(II) and 2-oxoglutarate-dependent dioxygenases, and specifically recognizes the m6A modification within a conserved binding pocket (1). ALKBH5 knockout mice have been shown to be viable but harbor defects in spermatogenesis, primarily due to rampant apoptosis in spermatocytes (2). Specifically, ALKBH5 is required for late meiotic and haploid phases of spermatogenesis, and loss of m6A removal impairs mRNA splicing and stability necessary for the expression of key meiotic genes (3). ALKBH5 expression has been shown to be elevated in hypoxic models of breast cancer, resulting in increased m6A demethylation of NANOG mRNA and consequently promoting increased NANOG protein expression and accumulation of breast cancer stem cells (4). Knockdown of ALKBH5 has been shown to impair tumor formation in MDA-MB-231 breast cancer cells, and inhibition of ALKBH5 also represses tumorigenesis in glioblastoma stem-like cells (4,5). Interestingly, ALKBH5 may also show promise as a novel biomarker for pancreatic cancer, where increased expression was associated with higher overall survival rates (6).

  1. Xu, C. et al. (2014) J Biol Chem 289, 17299-311.
  2. Zheng, G. et al. (2013) Mol Cell 49, 18-29.
  3. Tang, C. et al. (2018) Proc Natl Acad Sci U S A 115, E325-E333.
  4. Zhang, C. et al. (2016) Proc Natl Acad Sci U S A 113, E2047-56.
  5. Zhang, S. et al. (2017) Cancer Cell 31, 591-606.e6.
  6. Cho, S.H. et al. (2018) Ann Hepatobiliary Pancreat Surg 22, 305-9.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 5
#49015

ALKBH5 (E3F6E) Rabbit mAb

Western Blotting Image 1: ALKBH5 (E3F6E) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using ALKBH5 (E3F6E) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: ALKBH5 (E3F6E) Rabbit mAb Expand Image
Simple Western analysis of lysates (0.1 mg/mL) from MCF-7 cells using ALKBH5 (E3F6E) Rabbit mAb #49015. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess ​​​​​​​Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: ALKBH5 (E3F6E) Rabbit mAb Expand Image
Immunoprecipitation of ALKBH5 protein from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ALKBH5 (E3F6E) Rabbit mAb. Western blot analysis was performed using ALKBH5 (E3F6E) Rabbit mAb. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used as a secondary antibody.
Immunofluorescence Image 1: ALKBH5 (E3F6E) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HCT 116 cells, mock transfected (left, high-expressing) or transfected with siRNA directed against ALKBH5 (right, low-expressing), using ALKBH5 (E3F6E) Rabbit mAb (green), DyLight 554 Phalloidin #13054 (red), and DAPI #4083 (blue).