Revision 6

#14145Store 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-F, IF-IC

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

30

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q00535

Entrez-Gene Id:

1020

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunofluorescence (Frozen) 1:200 - 1:400
Immunofluorescence (Immunocytochemistry) 1:200 - 1:400

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

CDK5 (D1F7M) Rabbit mAb recognizes endogenous levels of total CDK5 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro253 of human CDK5 protein.

Background

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that are activated by cyclins and govern eukaryotic cell cycle progression. While CDK5 shares high sequence homology with its family members, it is thought mainly to function in postmitotic neurons to regulate the cytoarchitecture of these cells. Analogous to cyclins, the regulatory subunits p35 and p39 associate with and activate CDK5 despite the lack of sequence homology. CDK5 is ubiquitously expressed, with high levels of kinase activity detected primarily in the nervous system due to the narrow expression pattern of p35 and p39 in post-mitotic neurons. A large number of CDK5 substrates have been identified although no substrates have been specifically attributed to p35 or p39. Substrates of CDK5 include p35, PAK1, Src, β-catenin, tau, neurofilament-H, neurofilament-M, synapsin-1, APP, DARPP32, PP1-inhibitor, and Rb. p35 is rapidly degraded (T1/2 <20 min) by the ubiquitin-proteasome pathway (1). However, p35 stability increases as CDK5 kinase activity decreases, likely as a result of decreased phosphorylation of p35 at Thr138 by CDK5 (2). Proteolytic cleavage of p35 by calpain produces p25 upon neurotoxic insult, resulting in prolonged activation of CDK5 by p25. Research studies have shown accumulation of p25 in neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS) (3,4).

  1. Dhavan, R. and Tsai, L.H. (2001) Nat Rev Mol Cell Biol 2, 749-59.
  2. Patrick, G.N. et al. (1998) J Biol Chem 273, 24057-64.
  3. Lee, M.S. et al. (2000) Nature 405, 360-4.
  4. Kusakawa, G. et al. (2000) J Biol Chem 275, 17166-72.

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-F: Immunofluorescence (Frozen) 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

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

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 6
#14145

CDK5 (D1F7M) Rabbit mAb

Western Blotting Image 1: CDK5 (D1F7M) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa cells expressing either shCDK5 or non-targeting shRNA (shNT), using CDK5 (D1F7M) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: CDK5 (D1F7M) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa using CDK5 (D1F7M) Rabbit mAb #14145. 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: CDK5 (D1F7M) Rabbit mAb Expand Image

Immunoprecipitation of CDK5 protein from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CDK5 (D1F7M) Rabbit mAb. Western blot analysis was performed using CDK5 (D1F7M) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

Immunofluorescence Image 1: CDK5 (D1F7M) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse retina labeled with CDK5 (D1F7M) Rabbit mAb #14145 (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with Myelin Basic Protein (D8X4Q) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #73004 (magenta pseudocolor) and DAPI #4083 (blue).
Immunofluorescence Image 2: CDK5 (D1F7M) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse cortex labeled with CDK5 (D1F7M) Rabbit mAb #14145 (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with Myelin Basic Protein (D8X4Q) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #73004 (magenta pseudocolor) and DAPI #4083 (blue).
Immunofluorescence Image 3: CDK5 (D1F7M) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse cerebellum labeled with CDK5 (D1F7M) Rabbit mAb #14145 (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with Myelin Basic Protein (D8X4Q) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #73004 (magenta pseudocolor).
Immunofluorescence Image 1: CDK5 (D1F7M) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of control HeLa cells (left) or HeLa cells expressing shCDK5 (right) using CDK5 (D1F7M) Rabbit mAb (green), Calumenin (4C6) Mouse mAb #11991 (red), and DAPI #4083 (blue).