Western blot analysis of Ubiquitin was performed on HeLa cells either left untreated (right panel) or treated with 10uM of MG132 proteasome inhibitor (left panel) for 2 hours at 37C. Whole cell lysates were prepared using IP Lysis buffer (Product # 87787), and 10ug of the lysates along with 5ul of PageRuler Plus Prestained Protein Ladder (Product # 26619) were loaded onto a Novex® 10-20% Tricine Gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. Ubiquitin was detected at ~8.5kDa after probing with a Ubiquitin polyclonal antibody (Product # PA1-187) at a dilution of 1:1000 in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: In addition to detecting ubiquitin monomer, this antibody also detects poly-ubiquitinated proteins at various molecular weights.
|Tested species reactivity||Dog, Human, Mouse, Non-human primate, Rat|
|Host / Isotype||Rabbit / IgG|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:100-1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-187 detects both the ubiquitin monomer as well as poly-ubiquitinated proteins of various molecular weights.
Ubiquitin is a conserved 76 amino acid polypeptide and can affect proteasomal degradation of the protein it is bound to, or mediate interactions with other proteins related to post-translational modifications. The degradation of cellular regulatory proteins by the Uubiquitin pathway is important as it controls the cellular growth and proliferation. Ubiquitin-dependent proteolysis occurs after a covalent attachment of the peptide to a lysine residue of a protein, which involves three enzymatic reactions: E1, E2 and E3. The first reaction involves ubiquitin-activating enzyme. The third reaction uses enzyme ubiquitin ligase (E3) to transfer the activated ubiquitin from E2 to a lysine residue on a protein, or directly transfers the ubiquitin from E2 to the substrate.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.