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8523
BAG6 Antibody
Primary Antibodies
Polyclonal Antibody

BAG6 Antibody #8523

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  1. WB
Western Blotting Image 1: BAG6 Antibody
Western blot analysis of extracts from various cell lines using BAG6 Antibody.
To Purchase # 8523S
製品番号 サイズ 価格 在庫
8523S
100 µl

Supporting Data

REACTIVITY H M R Mk Pg
SENSITIVITY Endogenous
MW (kDa) 150
SOURCE Rabbit

Application Key:

  • WB-Western Blot
  • 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
  • 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
  • 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 and 50% glycerol. 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 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

BAG6 Antibody recognizes endogenous levels of total BAG6 protein. It does not cross-react with other BCL2-associated athanogene (Bag) family members.

Species Reactivity:

Human, Mouse, Rat, Monkey, Pig

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human BAG6 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Background

BAG6 (BCL2-associated athanogene-6), alternately known as BAT3 (HLA-B-associated transcript 3), was originally identified as a gene within the class III region of the human major histocompatibility complex, but has subsequently been found to exhibit protein chaperone activity. BAG6, in conjunction with other chaperone proteins and ubiquitin ligases, regulates protein stability and insertion of tail-anchored membrane proteins into the endoplasmic reticulum (1-3). The BAT3 complex, consisting of BAG6, TRC35 and Ubl4a localizes to ribosomes synthesizing membrane proteins and facilitates tailed-anchored protein capture by TRC40 and subsequent insertion of the nascent protein in to the ER membrane (4,5). BAG6 also plays a critical role in clearing cells of mis-folded and mis-localized peptides via endoplasmic reticulum-associated degradation and the ubiquitin-proteasome system (1,6,7). BAG6 may also act as a chaperone for glycoproteins through its interaction with DERLIN2 (8). In addition to its role as a chaperone, BAG6 has also been implicated in regulating chromatin structure and gene expression. For example, BAG6 and SET1A act as binding partners for BORIS to effect changes of chromatin structure and gene expression (9). Similarly, increased expression of BAG6 induces p300-mediated acetylation of p53, which is required for DNA damage response (10). BAG6 has also been found to interact with TGF-β, and in so doing acts as a positive regulator of TGF-β1 stimulation of type 1 collagen expression (11). BAG6 also suppresses bone morphogenic protein (BMP) signaling via its interaction with and regulation of small C-terminal domain phosphatase (SCP) that dephosphorylates SMAD proteins resulting in subsequent termination of BMP-mediated events (12).
  1. Hessa, T. et al. (2011) Nature 475, 394-7.
  2. David, R. (2011) Nat Rev Mol Cell Biol 12, 550.
  3. Ast, T. and Schuldiner, M. (2011) Curr Biol 21, R692-5.
  4. Mariappan, M. et al. (2010) Nature 466, 1120-4.
  5. Leznicki, P. et al. (2010) J Cell Sci 123, 2170-8.
  6. Minami, R. et al. (2010) J Cell Biol 190, 637-50.
  7. Wang, Q. et al. (2011) Mol Cell 42, 758-70.
  8. Claessen, J.H. and Ploegh, H.L. (2011) PLoS One 6, e28542.
  9. Nguyen, P. et al. (2008) Mol Cell Biol 28, 6720-9.
  10. Sasaki, T. et al. (2007) Genes Dev 21, 848-61.
  11. Kwak, J.H. et al. (2008) J Biol Chem 283, 19816-25.
  12. Goto, K. et al. (2011) Cell Death Dis 2, e236.

Pathways & Proteins

Explore pathways + proteins related to this product.

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