Anti-SQSTM1 / p62 antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Instead of loading 20–30 μg of total protein as per the protocol, this article used 40ug protein

Protocol tips
Instead of loading 20–30 μg of total protein as per the protocol, this article used 40ug protein

Publication protocol

C6 cells were seeded in the six-well plates and then treated with AG-1031 for 24 h. Using lysis buffer and 1% PMSF to lyse the cells for 15 min on ice. Cell lysates were ultracentrifuged at 12000 rpm for 15 min at 4 °C, and then collecting the supernatant as the total cellular proteins. A BCA protein assay kit was used to determine the concentrations. Adding loading buffer to the supernatant (ratio is 4:1) and boiling it at 95-100 °C for 10 min. Samples (40 µg protein) were extracted from those cells, and were resolved to 10-13% SDS-PAGE and transferred to polyvinylidene
fluoride (PVDF) membranes. Blocking the membrane for 1 h subsequently and incubating it with primary antibody diluted of 1:1000 respectively during a night at 4°C. Probing the immunoreactivity by adding secondary antibody diluted to 1:5000 after washing the membrane with TBST and detecting it with chemiluminescent HRP detection kit. The protein transfer was confirmed by immunoblotting for
determination of actin protein using β-actin antibody
(1:1000) on the same Western blot.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - C6 using Anti-SQSTM1 / p62 antibody from Abcam.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - C6 using Anti-SQSTM1 / p62 antibody from Abcam. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.