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94350
NLK (D9X3C) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

NLK (D9X3C) Rabbit mAb #94350

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  1. WB
Western Blotting Image 1: NLK (D9X3C) Rabbit mAb

Western blot analysis of extracts from SW480, HCT 116 and C2C12 cells using NLK (D9X3C) Rabbit mAb.

To Purchase # 94350S
Product # Size Price
94350S
100 µl $ 268

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa) 58
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • 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
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

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.

Protocol

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Western Blotting Protocol

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

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883)by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 263

Specificity / Sensitivity

NLK (D9X3C) Rabbit mAb recognizes endogenous levels of total NLK protein.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

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

Background

Nemo-like kinase (NLK ) is a serine/threonine-protein kinase that regulates multiple signaling pathways, including Wnt/β-catenin, TGFβ, IL-6, and Notch (1-4). NLK contributes to cell proliferation, differentiation, cell fate determination during early embryogenesis and nervous system development in vertebrates (5-7). Recent studies showed that NLK is aberrantly expressed in various types of cancer where it regulates cancer cell proliferation, migration, invasion and survival (8-11). NLK is localized predominantly in nucleus and at a lower level in cytoplasm(12). Homodimerization of NLK is required for its activation and nuclear localization. NLK is activated via intermolecular autophosphorylation at Thr286 (13). NLK interacts with and phosphorylates a number of transcription factors including FOXO1, FOXO4, MYB, NOTCH1 and TCF7L2/TCF4, and LEF-1/TCF (14-18). NLK also associates with E3 ubiquitin ligase NARF and Raptor and regulates their function (19,20).

  1. Smit, L. et al. (2004) J Biol Chem 279, 17232-40.
  2. Ohkawara, B. et al. (2004) Genes Dev 18, 381-6.
  3. Kojima, H. et al. (2005) Proc Natl Acad Sci U S A 102, 4524-9.
  4. Ishitani, T. et al. (2010) Nat Cell Biol 12, 278-85.
  5. Hyodo-Miura, J. et al. (2002) Genes Cells 7, 487-96.
  6. Thorpe, C.J. and Moon, R.T. (2004) Development 131, 2899-909.
  7. Satoh, K. et al. (2007) Mol Cell Biol 27, 7623-30.
  8. Li, M. et al. (2013) Tumour Biol 34, 3995-4000.
  9. Lv, L. et al. (2014) J Cell Biochem 115, 81-92.
  10. Huang, Y. et al. (2013) PLoS One 8, e69148.
  11. Dong, J.R. et al. (2013) Asian Pac J Cancer Prev 14, 7137-41.
  12. Brott, B.K. et al. (1998) Proc Natl Acad Sci U S A 95, 963-8.
  13. Ishitani, S. et al. (2011) Mol Biol Cell 22, 266-77.
  14. Ishitani, T. et al. (2003) Mol Cell Biol 23, 1379-89.
  15. Kanei-Ishii, C. et al. (2004) Genes Dev 18, 816-29.
  16. Kim, S. et al. (2010) J Biol Chem 285, 8122-9.
  17. Togi, S. et al. (2011) J Biol Chem 286, 19170-7.
  18. Szypowska, A.A. et al. (2011) Antioxid Redox Signal 14, 563-78.
  19. Yamada, M. et al. (2006) J Biol Chem 281, 20749-60.
  20. Yuan, H.X. et al. (2015) Genes Dev 29, 2362-76.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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