1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Is unsuitable for Flow Cyt,ICC or IP.
Isoform 1 functions as extracellular chaperone that prevents aggregation of nonnative proteins. Prevents stress-induced aggregation of blood plasma proteins. Inhibits formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro). Does not require ATP. Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70. Does not refold proteins by itself. Binding to cell surface receptors triggers internalization of the chaperone-client complex and subsequent lysosomal or proteasomal degradation. Secreted isoform 1 protects cells against apoptosis and against cytolysis by complement. Intracellular isoforms interact with ubiquitin and SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes and promote the ubiquitination and subsequent proteasomal degradation of target proteins. Promotes proteasomal degradation of COMMD1 and IKBKB. Modulates NF-kappa-B transcriptional activity. Nuclear isoforms promote apoptosis. Mitochondrial isoforms suppress BAX-dependent release of cytochrome c into the cytoplasm and inhibit apoptosis. Plays a role in the regulation of cell proliferation.
Detected in blood plasma, cerebrospinal fluid, milk, seminal plasma and colon mucosa. Detected in the germinal center of colon lymphoid nodules and in colon parasympathetic ganglia of the Auerbach plexus (at protein level). Ubiquitous. Detected in brain, testis, ovary, liver and pancreas, and at lower levels in kidney, heart, spleen and lung.
Belongs to the clusterin family.
Isoform 1 is proteolytically cleaved on its way through the secretory system, probably within the Golgi lumen. Polyubiquitinated, leading to proteasomal degradation. Heavily N-glycosylated. About 30% of the protein mass is comprised of complex N-linked carbohydrate.
Secreted. Can retrotranslocate from the secretory compartments to the cytosol upon cellular stress and Nucleus. Cytoplasm. Mitochondrion membrane. Cytoplasm, cytosol. Microsome. Endoplasmic reticulum. Cytoplasmic vesicle, secretory vesicle, chromaffin granule. Isoforms lacking the N-terminal signal sequence have been shown to be cytoplasmic and/or nuclear. Secreted isoforms can retrotranslocate from the secretory compartments to the cytosol upon cellular stress. Detected in perinuclear foci that may be aggresomes containing misfolded, ubiquitinated proteins. Detected at the mitochondrion membrane upon induction of apoptosis.
Western blot - Anti-Clusterin antibody [EPR2911] (ab92548)
Predicted band size : 52 kDa
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: Clusterin knockout HAP1 whole cell lysate (20 µg) Lane 3: U87-MG whole cell lysate (20 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab92548 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab92548 was shown to recognize Clusterin in wild-type HAP1 cells as signal was lost at the expected MW in Clusterin knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Clusterin knockout samples were subjected to SDS-PAGE. Ab92548 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Clusterin antibody [EPR2911] (ab92548)This image is courtesy of an Abreview by Ruma Raha-Chowdhury.
ab92548 staining Apolipoprotein J in mouse brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, cut into 20 micron slices, permeablized with 0.1 M PBS with 1% Triton X and blocked with 10% serum for 60 minutes at 24°C. The sample was incubated with primary antibody (1/100 in 0.1M PBST with 10% donkeys serum) at 4°C for 24 hours. An Alexa Fluor® 488-conjugated donkey polyclonal (1/1000) was used as the secondary antibody.