Anti-SQSTM1 / p62 antibody

• Supplier: Abcam
• Catalog: ab56416
• Prices: $401.00
• Sizes: 100 µg
• Host: Mouse
• Clonality: Monoclonal
• Isotype: IgG2a
• Applications: IHC-P WB ICC/IF ICC/IF FC IHC-F
• Species Reactivities: Mouse, Rat, Human, Monkey, Hamster
• Antigen: Recombinant full length protein, corresponding to amino acids 1-441 of Human SQSTM1/ p62
• Description: Mouse Monoclonal
• Gene: SQSTM1
• Conjugate: Unconjugated

Product images

Western blot against tagged recombinant protein immunogen using ab56416 SQSTM1 / p62 antibody at 1ug/ml.

Monoclonal antibody to SQSTM1 (ab56416) on HeLa cell, antibody concentration 10 ug/ml.

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. See Abreview

All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilutionLane 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.SecondaryHorseradish peroxidase-conjugated secondary developed using the ECL techniqueImage from Vazquez-Martin A et al, PLoS One. 2009 Jul 16;4(7):e6251, Fig 4.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.

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.

All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilutionLane 1 : ControlLane 2 : Starved in HBSS for 2 hoursLane 3 : Starved in HBSS for 4 hoursLane 4 : Starved in HBSS for 8 hoursLane 5 : Starved in HBSS 8 hours + 200 nM Baf A1Lane 6 : Starved in HBSS 8 hours + 4 uM Mg132Lysates/proteins at 40 µg per lane.SecondaryHRP conjugated goat anti-mouse polyclonal at 1/5000 dilutiondeveloped using the ECL techniqueObserved band size : 62 kDa (why is the actual band size different from the predicted?)Exposure time : 10 secondsImage courtesy of Dr Randal Tibbetts by Abreview.All lanes are whole cell lysate prepared from HeLa cells. Treatments are listed.See Abreview

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 DyLight® 488 goat anti-mouse IgG (H+L) (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.

developed using the ECL techniquePerformed under reducing conditions.Observed band size : 62 kDa (why is the actual band size different from the predicted?)Exposure time : 2 secondsThis image is courtesy of an anonymous AbreviewWestern blot analysis of Rhesus monkey retinal pigmented epithelium whole cell lysate (20µg/lane) treated with increasing concrentration of an autophagy inhibitor. SQSTM1 / p62 was labelling with ab56416 at 1/1000. An Alkaline Phosphatase-conjugated rabbit anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.See Abreview

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