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11864
PSMA2 (D3A4) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

PSMA2 (D3A4) Rabbit mAb #11864

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  1. WB
Western Blotting Image 1 - PSMA2 (D3A4) Rabbit mAb

Western blot analysis of extracts from various cell lines using PSMA2 (D3A4) Rabbit mAb.

Western Blotting Image 2 - PSMA2 (D3A4) Rabbit mAb

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMA1 (hPSMA1-Myc/DDK; +), full-length human PSMA2 (hPSMA2-Myc/DDK; +), full-length human PSMA3 (hPSMA3-Myc/DDK; +), full-length human PSMA4 (hPSMA4-Myc/DDK; +), full-length human PSMA6 (hPSMA6-Myc/DDK; +), or full-length human PSMA7 (hPSMA7-Myc/DDK; +), using PSMA2 (D3A4) Rabbit mAb (upper) or DYKDDDDK Tag Antibody #2368 (lower).

To Purchase # 11864S
Product # Size Price
11864S
100 µl $ 268

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 25
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 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. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  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 (#13953, 5 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

PSMA2 (D3A4) Rabbit mAb recognizes endogenous levels of total PSMA2 protein. This antibody does not cross-react with other α subunits of the 20S proteasome.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology:

Chicken, Xenopus, Zebrafish, Bovine, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe173 of human PSMA2 protein.

Background

The 20S proteasome is the major proteolytic enzyme complex involved in intracellular protein degradation. It consists of four stacked rings, each with seven distinct subunits. The two outer layers are identical rings composed of α subunits (called PSMAs), and the two inner layers are identical rings composed of β subunits. While the catalytic sites are located on the β rings (1-3), the α subunits are important for assembly and as binding sites for regulatory proteins (4). Seven different α and ten different β proteasome genes have been identified in mammals (5). PA700, PA28, and PA200 are three major protein complexes that function as activators of the 20S proteasome. PA700 binds polyubiquitin with high affinity and associates with the 20S proteasome to form the 26S proteasome, which preferentially degrades poly-ubiquitinated proteins (1-3). The proteasome has a broad substrate spectrum that includes cell cycle regulators, signaling molecules, tumor suppressors, and transcription factors. By controlling the degradation of these intracellular proteins, the proteasome functions in cell cycle regulation, cancer development, immune responses, protein folding, and disease progression (6-9).

  1. Dahlmann, B. (2005) Essays Biochem. 41, 31-48.
  2. Pickart, C.M. and Cohen, R.E. (2004) Nat. Rev. Mol. Cell Biol. 5, 177-187.
  3. Nandi, D. et al. (2006) J. Biosci. 31, 137-155.
  4. Lupas, A. et al. (1993) Enzyme Protein 47, 252-273.
  5. Monaco, J.J. and Nandi, D. (1995) Annu. Rev. Genet. 29, 729-754.
  6. Murray, A.W. (2004) Cell 116, 221-234.
  7. Ciechanover, A. (2006) Proc. Am. Thorac. Soc. 3, 21-31.
  8. Wang, J. and Maldonado, M.A. (2006) Cell. Mol. Immunol. 3, 255-261.
  9. Rubinsztein, D.C. (2006) Nature 443, 780-786.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

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