Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62

Autophagy assay cell type - N27 dopaminergic cells

Experiment
Autophagy assay cell type - N27 dopaminergic cells
Product
Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62 from BD Biosciences
Manufacturer
BD Biosciences

Protocol tips

Protocol tips
- Dilute Ab at 1:500

- Incubate Ab for 1 h at room temperature

Publication protocol

Western-blotting
Following treatments, cells were processed as described previously [2]. Basically, cells were washed with PBS 1X and lysed in sample buffer (SB) 1X (2% (v/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, and 50 mM Tris–HCl, pH 6.8, in distilled water) by pipetting until homogenization. Protein concentration was measured based on the bicinchoninic acid assay, using bovine serum albumin as a standard. Samples were heated at 95 °C for 10 min before their quantification.

Equal amounts of protein (25–40 µg/condition) were resolved by 12% SDS-gel electrophoresis and transferred to polyvinylidene fluoride membranes, according to a partially modified conventional protocol [3]. Immunodetection included transferring (15 V during 15 min, per each membrane) and blocking of the membrane with WB blocking solution (10% w/v fat free milk in Tris-buffered saline with Tween 20 (TBST)) for 1 h at room temperature. After washing the membranes 2 times with TBST 1X, blots were incubated with the corresponding primary antibody: p62/SQSTM1 (1:5000) (BD Transduction Laboratories, 610498), LC3-B (1:5000) (Sigma–Aldrich, L7543) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000) (Millipore, MAB374) at 4 °C overnight, GAPDH (1 h at room temperature). The membranes were washed 2 times with TBST 1X and subsequently incubated with their respective HRP-conjugated secondary antibodies (1:10000) (Bio-Rad, 170–6515 and 170-5047 for rabbit and mouse antibodies, respectively) for 1 h at room temperature. Detection of bound antibodies was visualized by chemiluminescence using ECL substrate (Thermo Scientific, 32106). Quantification data analysis was performed using ImageJ software (NIH), establishing GAPDH protein levels as a loading control.

Immunofluorescence
For the detection of endogenous p62 and LC3B, cells were seeded on coverslips, fixed with paraformaldehyde (4% in PBS 1X) and permeabilized with Triton X-100 solution (0.1% in PBS 1X) for 10 min. To block non-specific binding, cells were incubated with 10% FBS in PBS 1X for 20 min, followed by incubation with primary antibodies anti-p62 (1:500) and anti-LC3B (1:500) for 1 h at room temperature. After that, cells were incubated with Alexa Fluor 488 anti-rabbit (1:1000) (Molecular Probes, A-11034) and 568 anti-mouse (1:1000) (Molecular Probes, A-11004) secondary antibodies for LC3 and p62, respectively. Finally, coverslips were mounted on microscope slides, by using fluoromount-G (SouthernBiotech, 0100–01) medium. Images were taken by using an inverted fluorescence microscope (Olympus, IX-51) equipped with a camera (Olympus, DP70). The quantitative measurement of the fluorescence signal was performed using ImageJ software analyzing at least 200 cells per condition. Immunofluorescence procedure was developed as previously described [2].

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

Reviews

Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62 from BD Biosciences has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - N27 dopaminergic cells using Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62 from BD Biosciences.

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

Manufacturer protocol

Download the product protocol from BD Biosciences for Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - N27 dopaminergic cells using Purified Mouse Anti-Nucleoporin p62 Clone 53/Nucleoporin p62 from BD Biosciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Become shareholder Discussions About us Contact Privacy Terms