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REACTIVITY SENSITIVITY MW (kDa)
H M R Mk Endogenous 22

Western blot analysis of extracts from various cell lines using PSMB5 Antibody.

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Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMB5 (hPSMB5-Myc/DDK; +) and Myc/DDK-tagged full-length human PSMB8 (hPSMB8-Myc/DDK; +), using PSMB5 Antibody (upper) or DYKDDDDK Tag Antibody #2368 (lower).

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Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 72hr; +), using PSMB5 Antibody (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

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

A. Solutions and Reagents

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. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. 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 (#7720, 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 datasheet) 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 November 2013

protocol id: 263

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

PSMB5 Antibody recognizes endogenous levels of total PSMB5 protein. Based upon sequence alignment, this antibody is predicted to react with precursor and mature forms of PSMB5. This antibody does not cross-react with PSMB8.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PSMB5 protein. Antibodies are purified by protein A and peptide affinity chromatography.

The 26S proteasome is a highly abundant proteolytic complex involved in the degradation of ubiquitinated substrate proteins. It consists largely of two sub-complexes, the 20S catalytic core particle (CP) and the 19S/PA700 regulatory particle (RP) that can cap either end of the CP. The CP consists of two stacked heteroheptameric β-rings (β1-7) that contain three catalytic β-subunits and are flanked on either side by two heteroheptameric α-rings (α1-7). The RP includes a base and a lid, each having multiple subunits. The base, in part, is composed of a heterohexameric ring of ATPase subunits belonging to the AAA (ATPases Associated with diverse cellular Activities) family. The ATPase subunits function to unfold the substrate and open the gate formed by the α-subunits, thus exposing the unfolded substrate to the catalytic β-subunits. The lid consists of ubiquitin receptors and DUBs that function in recruitment of ubiquitinated substrates and modification of ubiquitin chain topology (1,2). Other modulators of proteasome activity, such as PA28/11S REG, can also bind to the end of the 20S CP and activate it (1,2).


The core particle performs three types of catalytic activities inside its chamber: chymotrypsin-like, trypsin-like, and caspase-like activities, which are provided by the constitutively expressed PSMB5 (β5/MB1/X/LMPX/Macropain epsilon chain), PSMB7 (β2/Z/Macropain chain Z) and PSMB6 (β1/Y/LMPY/Macropain delta chain) subunits, respectively. These catalytic subunits belong to the N-terminal nucleophile (Ntn) hydrolase family and are characterized by an unusual, essentially single-residue active site: The N-terminal threonine of each proteolytic subunit provides both the catalytic nucleophile (on its side chain) and the primary proton acceptor (on the main chain N-terminus). The catalytic β-subunits are synthesized with N-terminal propeptides, which are removed at the final step of proteasome biogenesis by limited proteolysis to expose the catalytic threonine residues (3). In immune responsive cells the constitutively expressed PSMB6, PSMB7, and PSMB5 subunits are replaced by three highly homologous induced β-subunits: PSMB9 (β1i/LMP2/RING12), PSMB10 (β2i/MECL-1/LMP10) and PSMB8 (β5i/LMP7/RING10), respectively, to form the immunoproteasome that has higher chymotrypsin-like and trypsin-like activities known to be favorable for antigen processing (4,5). PSMB5 is downregulated at the protein level by IFN-γ and replaced by PSMB8 in order to remodel the proteolytic specificity of the proteasome for more appropriate immunological processing of endogenous antigens (6-8). PSMB5 is also one of the predominant targets of bortezomib, an inhibitor of the chymotrypsin-like activity of the proteasome (9).


1.  Finley, D. (2009) Annu Rev Biochem 78, 477-513.

2.  Lee, M.J. et al. (2011) Mol Cell Proteomics 10, R110.003871.

3.  Murata, S. et al. (2009) Nat Rev Mol Cell Biol 10, 104-15.

4.  Boes, B. et al. (1994) J Exp Med 179, 901-9.

5.  Cardozo, C. and Kohanski, R.A. (1998) J Biol Chem 273, 16764-70.

6.  Akiyama, K. et al. (1994) Science 265, 1231-4.

7.  Akiyama, K. et al. (1994) FEBS Lett 343, 85-8.

8.  Gaczynska, M. et al. (1996) J Biol Chem 271, 17275-80.

9.  Oerlemans, R. et al. (2008) Blood 112, 2489-99.


Entrez-Gene Id 5693
Swiss-Prot Acc. P28074


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