View Featured Offers >>
78568
ATAD2 (E8Y2K) Rabbit mAb
Primary Antibodies
Monoclonal Antibody
  1. Products
  2. Primary Antibodies
  3. ATAD2 (E8Y2K) Rabbit mAb
R
Recombinant

ATAD2 (E8Y2K) Rabbit mAb #78568

Citations (2)
Filter:
  1. WB
  2. IHC
Western blot analysis of extracts from T-47D, K-562, and HCC1806 cells using ATAD2 (E8Y2K) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human urothelial carcinoma using ATAD2 (E8Y2K) Rabbit mAb (left) or ATAD2 Rabbit mAb (right). These two antibodies detect independent, unique epitopes on human ATAD2. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using ATAD2 (E8Y2K) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using ATAD2 (E8Y2K) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using ATAD2 (E8Y2K) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded T-47D cell pellet (left, high-expressing) or MCF 10A cell pellet (right, low-expressing) using ATAD2 (E8Y2K) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human gastrointestinal stromal tumor using ATAD2 (E8Y2K) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human testis using ATAD2 (E8Y2K) Rabbit mAb.
Image Gallery
Learn more about how we get our images
To Purchase # 78568
Cat. # Size Qty. Price
78568S		 100 µl

Carrier-Free Bulk & Custom Formulation

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 180
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species 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

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunohistochemistry (Paraffin) 1:200 - 1:800

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

PRINT

View >Collapse >

Western Blotting Protocol

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.

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

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

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. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. 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 (#59329, 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: 10

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

DETECTION REAGENT/SUBSTRATE COMPATIBILITY
RECOMMENDED
DETECTION REAGENTS		 SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653
COMPATIBLE
CHROMOGEN		 SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824
SignalStain® Deep Black Peroxidase Substrate Kit #72986  
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644  

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.


posted February 2010

revised June 2020

Protocol Id: 283

Specificity / Sensitivity

ATAD2 (E8Y2K) Rabbit mAb recognizes endogenous levels of total ATAD2 protein. Note: Non-specific non-nuclear staining was observed in brain in immunohistochemistry testing.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu910 of human ATAD2 protein.

Background

ATPase family AAA domain containing protein 2 (ATAD2) is an oncogenic protein that was originally identified as a coactivator for estrogen receptor (ESR1), and later identified as a coactivator for other transcription factors including c-Myc and E2F1, E2F2, and E2F3 proteins (1-4). ATAD2 is highly expressed and associated with poor prognosis in many types of cancer, including breast, uterine, colon, ovarian, stomach, non-small cell lung carcinoma, osteosarcoma, and cervical cancer (1,5-14). In cancer cells, overexpressed ATAD2 interacts with transcription factors and chromatin modifier proteins to induce the expression of genes that promote cell proliferation and inhibit apoptosis, ultimately promoting tumor growth (15,16). Indeed, knockdown of ATAD2 in pancreatic cancer cell lines has been shown to promote apoptosis, limit cell migration and invasion, and inhibit anchorage-independent growth (17). ATAD2 is a member of the ATPases associated with various cellular activities (AAA) family of proteins and contains a functional AAA domain in its central region, as well as a bromodomain near the C-terminus. The bromodomain binds to acetylated lysine residues on histone proteins, targeting ATAD2 protein to areas of active transcription, where it modulates chromatin structure and recruits additional transcription factors (18,19). Current efforts are underway to better characterize and develop inhibitors to the ATAD2 bromodomain for the treatment of various cancers (16,20-23).

  1. Zou, J.X. et al. (2007) Proc Natl Acad Sci U S A 104, 18067-72.
  2. Zou, J.X. et al. (2009) Cancer Res 69, 3339-46.
  3. Revenko, A.S. et al. (2010) Mol Cell Biol 30, 5260-72.
  4. Ciró, M. et al. (2009) Cancer Res 69, 8491-8.
  5. Wang, Y. et al. Lancet 365, 671-9.
  6. Teschendorff, A.E. et al. (2006) Genome Biol 7, R101.
  7. Fellenberg, J. et al. (2007) Mod Pathol 20, 1085-94.
  8. Petroziello, J. et al. (2004) Oncogene 23, 7734-45.
  9. De Angelis, P.M. et al. (2006) Mol Cancer 5, 20.
  10. Zheng, L. et al. (2015) Oncol Rep 33, 2337-44.
  11. Fouret, R. et al. (2012) Clin Cancer Res 18, 5606-16.
  12. Wan, W.N. et al. (2014) Asian Pac J Cancer Prev 15, 2777-83.
  13. Wu, G. et al. (2014) BMC Cancer 14, 107.
  14. Zhang, M. et al. (2016) Clin Transl Oncol 18, 776-81.
  15. Caron, C. et al. (2010) Oncogene 29, 5171-81.
  16. Boussouar, F. et al. (2013) Biochim Biophys Acta 1829, 1010-4.
  17. Liu, N. et al. (2019) Oncol Lett 17, 3489-94.
  18. Morozumi, Y. et al. (2016) J Mol Cell Biol 8, 349-62.
  19. Koo, S.J. et al. (2016) Oncotarget 7, 70323-35.
  20. Gay, J.C. et al. (2019) Proteins 87, 157-67.
  21. Hussain, M. et al. (2018) Expert Opin Ther Targets 22, 85-96.
  22. Bamborough, P. et al. (2018) J Med Chem 61, 8321-36.
  23. Zhou, Y. et al. (2018) Phys Chem Chem Phys 20, 23222-32.

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.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit our Trademark Information page.