P62/SQSTM1 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Tris-Tricine gels separate low MW proteins(less than 20 kDa) beer than Tris-Glycine gels. Gradient polyacrylamide gels can provide sharper bands and they separate a broader range of MW sizes on one gel, such as 10-500 kDa.	- Incubate primary and secondary antibodies for two hours at room temperature

Upstream tips
- Tris-Tricine gels separate low MW proteins(less than 20 kDa) beer than Tris-Glycine gels. Gradient polyacrylamide gels can provide sharper bands and they separate a broader range of MW sizes on one gel, such as 10-500 kDa.
Protocol tips
- Incubate primary and secondary antibodies for two hours at room temperature

Publication protocol

Protein extracts from culture cells and mice pancreatic tissues were separated and quantified by sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore). After blocking with 5% nonfat milk in phosphate buffer saline (PBS), membranes were immunoblotted with the indicated primary antibodies (including anti-LC3, anti-p62, antibeclin1, antiubiqutin, anti-phos-p65, anti-NFκB p65, anti-CaMKII and anti-phos-CaMKII bodies), and then with horseradish peroxidase-conjugated immunoglobulin G (IgG) secondary antibodies for two hours at room temperature. In this study, anti-α-tubulin antibody acted as the internal control. The resulting bands were visualized with an electrochemiluminescence (ECL) system (Amersham Biosciences).

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