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62224
Angiogenin (E8O6J) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Angiogenin (E8O6J) Rabbit mAb #62224

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  1. WB
Western Blotting Image 1: Angiogenin (E8O6J) Rabbit mAb
Western blot analysis of extracts from various cell lines using Angiogenin (E8O6J) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Negative expression of angiogenin protein in A2780 cells is consistent with the predicted expression pattern.
To Purchase # 62224
Cat. # Size Qty. Price
62224S
100 µl
$ 287

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 14
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
  • 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 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

Angiogenin (E8O6J) Rabbit mAb recognizes endogenous levels of total angiogenin protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to human angiogenin protein.

Background

Angiogenin (ANG, RNase 5) is a member of the ribonuclease A superfamily (1,2). It plays many diverse roles in cell and tissue function, including angiogenesis, innate immune function, signal transduction, and cellular stress responses. Angiogenin has been shown to promote tumor development, but has also been implicated in protection against neurodegenerative diseases (3-6). Angiogenin may be secreted into plasma, but it is also found within various tissues and cell types. It is a highly pleiotropic protein, acting through many diverse mechanisms. In the extracellular matrix, angiogenin promotes actin polymerization, and interacts with members of the plasminogen activation system to stimulate plasmin generation and promote cell migration (7,8). At the cell surface, angiogenin binds to receptors, such as plexin B2 and EGFR, activating signal transduction pathways (e.g., ERK and Akt) that promote cell proliferation and survival (9,10). Angiogenin also enters cells via receptor-mediated endocytosis; it may accumulate in nucleoli under growth conditions, or in the cytoplasm under stress conditions. Within nucleoli, angiogenin has been shown to epigenetically activate the promoter regions of rDNA, stimulating rRNA transcription. In conditions of cellular stress, angiogenin has been reported to translocate to the cytoplasm, and cleave tRNA in anticodon loops to produce tiRNA fragments. These stress-induced tiRNA fragments initiate cellular stress responses that serve to protect the host cell from apoptosis (11).
  1. Gupta, S.K. et al. (2013) Innate Immun 19, 86-97.
  2. Wang, Y.N. et al. (2018) J Biomed Sci 25, 83.
  3. Wiedłocha, A. (1999) Arch Immunol Ther Exp (Warsz) 47, 299-305.
  4. Tello-Montoliu, A. et al. (2006) J Thromb Haemost 4, 1864-74.
  5. Sarangdhar, M.A. and Allam, R. (2021) Int J Mol Sci 22, 1287. doi: 10.3390/ijms22031287.
  6. Prehn, J.H.M. and Jirström, E. (2020) Acta Pharmacol Sin 41, 442-446.
  7. Dutta, S. et al. (2014) Mol Oncol 8, 483-507.
  8. Pyatibratov, M.G. and Kostyukova, A.S. (2012) Int Rev Cell Mol Biol 295, 175-98.
  9. Yu, W. et al. (2017) Cell 171, 849-864.e25.
  10. Wang, Y.N. et al. (2018) Cancer Cell 33, 752-769.e8.
  11. Cucci, L.M. et al. (2021) Int J Mol Sci 22, 10704. doi: 10.3390/ijms221910704.

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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