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BNIP3 Antibody
Primary Antibodies
Polyclonal Antibody

BNIP3 Antibody #3769

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Citations (106)
  1. WB
Western Blotting Image 1: BNIP3 Antibody
Western blot analysis of extracts from C2C12 cells, untreated (-) or cobalt chloride-treated (100 μM; overnight) using BNIP3 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 2: BNIP3 Antibody
Western blot analysis of HeLa cells, mock transfected or transfected with mouse BNIP3, CTLL-2, RAW 264.7 and BaF3 cells using BNIP3 Antibody.
To Purchase # 3769
Cat. # Size Qty. Price
100 µl
$ 296

Supporting Data

MW (kDa) 22-28, 50-55

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&T-CUT&Tag
  • DB-Dot Blot
  • 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


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



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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

Specificity / Sensitivity

BNIP3 Antibody detects endogenous levels of total BNIP3 protein from mouse and rat.

Species Reactivity:

Mouse, Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser60 of BNIP3 from mouse/rat. Antibodies were purified by peptide affinity chromatography.


BNIP3 (Bcl-2/E1B-19kDa interacting protein 3) is a pro-apoptotic mitochondrial protein and Bcl-2 family member that contains a Bcl-2 homology 3 (BH3) domain and a carboxyl-terminal transmembrane (TM) domain (1-3). While BNIP3 has a predicted molecular weight of about 22 kDa, it runs anomalously on SDS-PAGE and includes a band of around 60 kDa that may be a dimeric form that is not reduced (2). BNIP3 associates with anti-apoptotic family members Bcl-2, Bcl-xL, and the adenovirus homologue E1B-19kDa. BNIP3 is distinct from other Bcl-2 family members that contain only the BH3 domain in that the TM domain, and not the BH3 domain, is required for mitochondrial targeting and pro-apoptotic activity (4). In addition to apoptosis, BNIP3 has been implicated in necrosis (5) and autophagy (6-11). In hypoxic conditions, BNIP3 can induce mitochondrial autophagy (mitophagy) by disrupting the Bcl-2-Beclin-1 complex (9). BNIP3 can also promote mitophagy by triggering the translocation of the E3 ubiquitin ligase Parkin to the mitochondria (10) or by directly binding LC3 on the autophagosome (11). BNIP3 may also localize to the endoplasmic reticulum (ER) where it can selectively induce the autophagic clearance of ER (ERphagy) (11). Increased expression of BNIP3 under hypoxic conditions is mainly regulated by the transcription factor HIF-1α (12-14). Silencing of the BNIP3 promoter by methylation has been observed in several types of cancer cells and may play an important role in their survival (14-18).
  1. Boyd, J.M. et al. (1994) Cell 79, 341-51.
  2. Chen, G. et al. (1997) J Exp Med 186, 1975-83.
  3. Yasuda, M. et al. (1998) J Biol Chem 273, 12415-21.
  4. Ray, R. et al. (2000) J Biol Chem 275, 1439-48.
  5. Vande Velde, C. et al. (2000) Mol Cell Biol 20, 5454-68.
  6. Daido, S. et al. (2004) Cancer Res 64, 4286-93.
  7. Tracy, K. et al. (2007) Mol Cell Biol 27, 6229-42.
  8. Quinsay, M.N. et al. (2010) Autophagy 6, 855-62.
  9. Bellot, G. et al. (2009) Mol Cell Biol 29, 2570-81.
  10. Lee, Y. et al. (2011) Am J Physiol Heart Circ Physiol 301, H1924-31.
  11. Hanna, R.A. et al. (2012) J Biol Chem 287, 19094-104.
  12. Bruick, R.K. (2000) Proc Natl Acad Sci USA 97, 9082-7.
  13. Guo, K. et al. (2001) Cell Death Differ 8, 367-76.
  14. Sowter, H.M. et al. (2001) Cancer Res 61, 6669-73.
  15. de Angelis, P.M. et al. (2004) Int J Oncol 24, 1279-88.
  16. Okami, J. et al. (2004) Cancer Res 64, 5338-46.
  17. Murai, M. et al. (2005) Clin Cancer Res 11, 1021-7.
  18. Murai, M. et al. (2005) Br J Cancer 92, 1165-72.

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