Anti-SQSTM1 / p62抗体- Autophagosome Marker (ab56416)

Lanes 1 - 4: Merged signal (red and green). Green - ab56416 observed at 55 kDa. Red - loading control, ab181602 observed at 37 kDa.  

 ab56416 was shown to react with SQSTM1 / p62 in HEK293T wildtype. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate ab263770) was used. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab56416 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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.

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ab56416 staining SQSTM1/p62 in H9C2 rat cardiomyocytes by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, 0.03% and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/100) in 1x TBS with 0.1% Blocking for 12 hours at 4°C. An Alexa Fluor^® 488-conjugated chicken polyclonal to mouse was used as secondary antibody.

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Blocked with 5% milk for 3 hours at 21°C.

Incubated with the primary antibody for 17 hours at 4°C.

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

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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. 

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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.

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All lanes are whole cell lysate prepared from HeLa cells. Treatments are listed.

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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

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