This antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to the N-Terminal region near amino acids 1-25 of Human Cul4A coupled to KLH.
Anti-Cul4A has been tested by western blot, immunoprecipitation, and immunohistochemistry. The antibody immunoprecipitates protein from cell lysates (using HeLa, U2OS and others). This antibody also co-immunoprecipitates at least 8 subunits of COP9, DDB1
Most modifiers mature by proteolytic processing from inactive precursors (a, amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC) -like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin. Data contributed by S.Jentsch, see references below.
Western Blot of Rabbit Anti-Cul4 Antibody. Lane 1: Opal Pre-Stained Ladder (p/n MB-210-0500). Lane 2: A431 whole cell lysate (p/n W09-000-361). Lane 3: U2OS whole cell lysate (p/n W09-001-374). Lane 4: HeLa whole cell lysate (p/n W09-000-364). Load: 35µg lysate per lane. Primary Antibody: Anti-Cul4 at 1:500 overnight at 2-8C. Secondary Antibody: Goat anti-rabbit IgG HRP (p/n 611-103-122) at 1:70,000 at RT for 30 mins. Expect: ~87.6kDa.
Immunohistochemistry. Rocklands anti-CUL4 antibody was diluted 1:500 to detect CUL4 in human testes tissue. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. The image shows the localization of antibody as the precipitated red signal, with a hematoxylin purple nuclear counter stain.
Western Blot of Rabbit Anti-Cul4 Antibody. Lane 1: Opal Pre-Stained Ladder (p/n MB-210-0500). Lane 2: human Testis. Lane 3: human skeletal muscle. Load: 35µg lysate per lane. Primary Antibody: Anti-Cul4 at 1:500 overnight at 2-8C. Secondary Antibody: Goat anti-rabbit IgG HRP (p/n 611-103-122) at 1:70,000 at RT for 30 mins. Expect: ~87.6kDa.
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