Anti-p62/SQSTM1 antibody produced in rabbit

Protocol tips

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	- incubate with primary antibodies at 4°C overnight.

Protocol tips
- incubate with primary antibodies at 4°C overnight.

Publication protocol

Cells lysates were prepared by homogenization in lysis buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% NP-40 [AppliChem, A1694], pH 7.5) with protease inhibitor cocktail tablets (Roche Diagnostics, Penzberg, Germany), and phosphatase inhibitor cocktail tablets (Thermo Scientific, 88667) for phosphorylation analysis. Protein lysates were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, ISEQ00010). Next, blots were blocked with 5% milk in Tris buffered saline/Tween20 buffer (TBST: 10 mM Tris, 150 mM NaCl, 0.1% Tween-20 [Sigma, P1379], pH 8.0) for 1 h and incubated with primary antibodies at 4°C overnight. After that, blots were briefly washed and incubated with secondary antibodies for another 1 h. After that, blots were visualized using a chemiluminescence kit (Bio-Rad, 170–5061). The densitometric analysis was performed using ImageJ software (National Institute of Health, Bethesda, MD, USA). ACTB, GAPDH and TUBB served as loading controls for different protein analyses.

To monitor the levels of soluble and insoluble SNCA fractions, cells were lysed using NP-40 buffer (10 mM HEPES, 142.5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% NP-40, pH 7.5) with protease inhibitor cocktail and rotated at 4°C for 1 h. Next, the lysates were centrifuged at 12,000 g for 10 min at 4°C. The supernatant fractions were then transferred to a new tube and mixed with 2*SDS buffer (12.5% 1 M Tris-HCl pH 6.8, 20% glycerine, 4% SDS [Genshare, 77–86–1]) to obtain the NP-40-soluble fraction. The sediments were subsequently washed with NP-40 buffer 3 times and mixed with 1*SDS buffer (6.25% 1 M Tris-HCl, pH 6.8, 10% glycerine, 2% SDS), followed by sonication to make sure that the sediment was completely lysed. After centrifugation, the supernatant fraction was then harvested and referred to as the NP-40-insoluble fraction.

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Manufacturer protocol

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