Cells were seeded onto 12-well plates. Soluble proteins were harvested using 2 × SDS lysis buffer (0.12 M Tris-Cl, pH 6.8, 3.3% SDS, 10% glycerol, 3.1% DTT). The lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; resolving buffer 1.5 M Tris-Cl, pH 8.8, stacking buffer 0.5 M Tris-Cl, pH 6.8). The gels were transferred to polyvinylidene difluoride membranes (BioRad). Incubate membrane with diluted antibody (anti-p62 (610833, BD Biosciences), anti-SQSTM1/p62 (5114s, CST), anti-LC3B (2775s, CST), anti-PARP (9542s, CST), anti-Caspase3 (9662s, CST), anti-Caspase9 (9502s, CST), anti-actin (ab6276, Abcam)) in 3% (w/v) skim milk or 5% (w/v) BSA overnight at 4°C. Rabbit and mouse secondary antibodies (1:3000 v/v, GE Healthcare) were treated in 3% (w/v) skim milk for 1 h at 25°C. Immunolabeling was detected using an ECL substrate (BioRad) according to the manufacturer’s instructions and a ChemiDoc XRS+ imaging system (BioRad).
Anti p62 610833
Manufactured by BD
Anti-p62 (610833) is a laboratory reagent used in research applications. It is an antibody that specifically recognizes the p62 protein, which plays a role in cellular processes such as autophagy and protein aggregation. The antibody can be used to detect and quantify the presence of p62 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ubiquitin 3936, by Cell Signaling Technology (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab6276, by Abcam (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Ultramicrotome, by Leica (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Ht7700 electron microscope, by Hitachi (1 mentions)
Anti ub sc 8017, by Santa Cruz Biotechnology (1 mentions)
Anti lc3 pm036, by MBL Life Science (1 mentions)
4 protocols using anti p62 610833
1
Quantitative Western Blot Analysis
2
Skin Biopsy Immunohistochemical Analysis
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Skin biopsy specimens were obtained 10 cm above the lateral malleolus. Immunohistochemical samples were fixed in 10% formalin, embedded in paraffin and sectioned at 6 mm thickness; this was followed by staining with H&E, anti-ubiquitin (3936; Cell Signaling) and anti-p62 (610833; BD Biosciences) antibodies.
3
Detailed Skin Biopsy Protocol for Immunohistochemistry and Electron Microscopy
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Skin biopsies were performed after local anesthesia. A 5-mm-diameter biopsy specimen was obtained at 10 cm above the lateral malleolus of the affected individual. For immunohistochemistry, all samples were fixed in 10% formalin and then were embedded in paraffin and sectioned at 6 mm thickness. Sections of all samples were stained by hematoxylin & eosin (H&E) and immunohistochemical analysis was performed. Anti-ubiquitin (3936; Cell Signaling) and anti-p62 (610833; BD Biosciences) antibodies were used for ubiquitin and p62 staining. Images were acquired by deconvolution digital microscope (Axioplan 2; Carl Zeiss).
Samples for electron microscopy were sliced into 1×1×3 mm3 size and fixed in 2.5% glutaraldehyde solution with Millonig’s phosphate buffer (pH 7.3). In the following preparations, the samples were incubated in 1% osmium tetroxide, then dehydrated with graded acetone, and embedded with resin. 50–100 nm ultrathin sections were prepared with an ultramicrotome (Leica Microsystems) and a diamond knife. After 3% uranyl acetate and lead nitrate double staining, prepared sections were examined and photographed on a Hitachi HT-7700 electron microscope.
Samples for electron microscopy were sliced into 1×1×3 mm3 size and fixed in 2.5% glutaraldehyde solution with Millonig’s phosphate buffer (pH 7.3). In the following preparations, the samples were incubated in 1% osmium tetroxide, then dehydrated with graded acetone, and embedded with resin. 50–100 nm ultrathin sections were prepared with an ultramicrotome (Leica Microsystems) and a diamond knife. After 3% uranyl acetate and lead nitrate double staining, prepared sections were examined and photographed on a Hitachi HT-7700 electron microscope.
4
Comprehensive Antibody Assay Protocol
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Assays were performed as described previously62 (link). S6K, pS6K, CREB, tubulin, HA and Flag antibodies were as previously described62 (link). Other antibodies were purchased as follows: anti-LC3 (PM036), MBL International; anti-P62 (610833), BD Biosciences; anti-AMPKα (2532S), anti-pAMPKα (2535S), anti-ULK1 (8054S), anti-pULK1 (Ser555) (5869S), anti-ERK (4695S), anti-pERK (4370S), anti-PLCγ1 (5690P), anti-pPLCγ1 (Tyr783) (14008S), Cell Signaling Technology; anti-TFEB (A303-673A), anti-InsP3R1 (A302-158A), Bethyl Laboratories; anti-PPP1CA (AYA361), anti-PPP1CB (AYA1025), anti-PPP1CC (AYA1169), ayaBIO; anti-calcineurin (A1063), ABclonal Technology; anti-FGFR1 (GTX107393), anti-β-Klotho (GTX45558), anti-InsP3R2 (GTX54772), GeneTex; anti-ASB3 (AP16752A), Abgent; anti-LAMP1 (L1418), anti-MID1 (M2198), Sigma-Aldrich; anti-UBR5 (ab134089), Abcam; anti-GFP (MMS-118P), Covance; anti-Ub (SC-8017), anti-PPP2CA (SC-80665), anti-DREAM (SC-9142), Santa Cruz.
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