Anti-SQSTM1 / p62 antibody (ab56416)

Protocol tips

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	For WB, incubate the primary Ab at overnight 4°C and for Immunofluorescence Microscopy incubate for 1h at RT

Protocol tips
For WB, incubate the primary Ab at overnight 4°C and for Immunofluorescence Microscopy incubate for 1h at RT

Publication protocol

Western Blotting
Whole cell lysates used for immunoblotting were prepared in lysis buffer containing a phosphatase inhibitor (Roche, 4693116001) on ice. Lysates were mixed with SDS loading buffer and boiled for 10 min. Protein samples were separated on SDS-PAGE gels and then transferred to PVDF membrane. Membranes were blocked by 5% nonfat milk (dissolved in PBST) for 1 h at room temperature. Corresponding primary antibody was used for overnight incubation at 4°C, followed by HRP-labeled secondary antibodies' incubation at room temperature for 2 hrs. For loading controls, membranes were probed with the antibody against β-actin or Gapdh. Densitometric ratios were quantified by ImageJ software.

Immunofluorescence Microscopy
Cells were grown to 60% confluence on a coverslip. After treatment, the cells were washed in PBS and fixed with 4% paraformaldehyde for 20 mins. After washed twice in PBS, the cells were permeabilized with 0.1% Triton X-100 (in PBS). Coverslips were blocked in 1% albumin bovine V (in PBS) for 30 mins at room temperature and then were incubated with primary antibody diluted in PBS (0.01% Triton X-100) for 1 h at room temperature. After washed with PBS for 4 × 5 mins, secondary antibodies were applied. Cell images were captured with a TCS SPF5 II Leica confocal microscope and software (LAS-AF-Lite_2.2.0_4758, Germany).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-SQSTM1 / p62 antibody (ab56416) below.

Download manufacturer protocol

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