The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 - 5 µg/ml.
Use a concentration of 10 µg/ml. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS). Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.
Use 0.5µg for 106 cells.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration. PubMed: 22577215
Adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. May play a role in titin/TTN downstream signaling in muscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates the interaction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels.
Involvement in disease
Defects in SQSTM1 are a cause of Paget disease of bone (PDB) [MIM:602080]. PDB is a metabolic bone disease affecting the axial skeleton and characterized by focal areas of increased and disorganized bone turn-over due to activated osteoclasts. Manifestations of the disease include bone pain, deformity, pathological fractures, deafness, neurological complications and increased risk of osteosarcoma. PDB is a chronic disease affecting 2 to 3% of the population above the age of 40 years.
The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinated substrates. Mediates the interaction with TRIM55. The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5 and NBR1. The ZZ-type zinc finger mediates the interaction with RIPK1.
Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN.
Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes to the sarcomeric band (By similarity). Localizes to late endosomes. May also localize to the nucleus. Accumulates in neurofibrillary tangles and in Lewy bodies of neurons from individuals with Alzheimer and Parkinson disease respectively. Enriched in Rosenthal fibers of pilocytic astrocytoma. In liver cells, accumulates in Mallory bodies associated with alcoholic hepatitis, Wilson disease, indian childhood cirrhosis and in hyaline bodies associated with hepatocellular carcinoma.
Monoclonal antibody to SQSTM1 (ab56416) on HeLa cell, antibody concentration 10 ug/ml.
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab56416)This image is courtesy of an Abreview submitted by Dr Alejandro Vazquez-Martin
ab56416 staining SQSTM1/p62 in Human A431 epidermoid cancer cells by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with formaldehyde, permeabilized with Triton X-100 and blocked with 5% BSA for 30 minutes at room temperature. Samples were incubated with primary antibody (1/50) in 5% BSA for 1 hour. An Alexa Fluor® 488-conjugated Goat monoclonal to mouse IgG (1/50) was used as secondary antibody.
Overlay histogram showing HeLa cells stained with ab56416 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56416, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
ab56416 (1µg/ml) staining SQSTM1 in human lymph node using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and cytoplasmic staining. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
Western blot - Anti-SQSTM1 / p62 antibody (ab56416)Image courtesy of Dr Randal Tibbetts by Abreview.
All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilution
Lane 1 : Control Lane 2 : Starved in HBSS for 2 hours Lane 3 : Starved in HBSS for 4 hours Lane 4 : Starved in HBSS for 8 hours Lane 5 : Starved in HBSS 8 hours + 200 nM Baf A1 Lane 6 : Starved in HBSS 8 hours + 4 uM Mg132
Lysates/proteins at 40 µg per lane.
Secondary All lanes : HRP conjugated goat anti-mouse polyclonal at 1/5000 dilution
Western blot - Anti-SQSTM1 / p62 antibody (ab56416)Image from Vazquez-Martin A et al, PLoS One. 2009 Jul 16;4(7):e6251, Fig 4.
All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilution
Lane 1 : Whole cell lysates prepared from Tzb-naive SKBR3 parental cells. Lane 2 : Whole cell lysates prepared from Tzb-refractory TzbR POOL1 cells. Lane 3 : Whole cell lysates prepared from Tzb-refractory TzbR POOL2 cells.
Lysates/proteins at 50 µg per lane.
Secondary All lanes : Horseradish peroxidase-conjugated secondary
Developed using the ECL technique.
Cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton® X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonylfluoride, and complete protease inhibitor cocktail for 30 minutes on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 minutes at 14,000×g, 4°C). Protein content was determined against a standardized control using the Pierce Protein Assay Kit. Equal amounts of protein were resuspended in 5× Laemmli sample buffer (10 minutes at 70°C), resolved by electrophoresis on 10% SDS-PAGE, and transferred onto nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 hour at room temperature with TBS-T buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] containing 5% (w/v) nonfat dry milk. The treated filters were washed in TBS-T and then incubated with the primary
Jin Q et al. DUSP1 alleviates cardiac ischemia/reperfusion injury by suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK pathways. Redox Biol14:576-587 (2018).
Read more (PubMed: 29149759) »
Mizuno Y et al. DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma. Sci Rep8:106 (2018).
Read more (PubMed: 29311600) »