Anti-Beclin 1 antibody (ab62557)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Protocol tips
If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Publication protocol

Immunofluorescence analysis
For immunofluorescence (IF) analysis, rPT cells were seeded on sterile cover slips placed in 24-well plates at a density of 2 × 105 cells/well. After treatments, rPT cells were fixed with 4% PFA, permeabilized by exposure for 10 min to 0.5% TritonX-100 and placed in blocking solution (5% BSA) for 1 h. Cells were then exposed to primary antibodies overnight at 4 °C, followed by incubation with fluorecscent secondary antibodies at room temperature for 1 h in the dark. Cells were washed three times with ice cold PBS, were then fixed on slides with 50% glycerin, and were observed under a laser scanning confocal microscope (TCS SP8 STED; Wetzlar, Hessen, GER). Images for colocalization analysis were assessed using the JaCoP plugin in ImageJ after thresholding of individual frames. All colocalization calculations were performed on three independent experiments with 50 cells 1 per condition in each experiment47.

Co-immunoprecipitation assay
rPT cells were harvested by Accutase™ Cell Detachment Solution. After cells were washed twice with ice cold PBS and sonicated, cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatant was collected and the total protein concentration (>400 μg per sample) was adjusted to 1 μg/μl. Protein lysates were incubated with primary antibodies overnight at 4 °C in a roto-shaker. 50 μl of Protein G SureBeads (Bio Rad) was add into the complexes and incubated in a roto-shaker for 2 h at room temperature. The Protein G complexes were washed three times with PBST on a magnetic frame. After washes, samples were eluted with 20 μl glycine elution buffer (20 mmol/L, pH 2.0) and 2 μl phosphate buffer (1 mmol/L, pH7.4). The eluate was resuspended in lysis buffer and boiled for 5 min to prepare for western blot analysis.

Western blot analysis
After cell fraction preparation and protein quantification with a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China), equal amounts of protein were separated by 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22-μm or 0.45-μm polyvinylidene difluoride (PVDF) membranes. After transfer, membranes were blocked in 5% skim milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the relevant primary antibodies. Membranes were then incubated with the appropriate secondary antibodies (1:5000) for 1 h at room temperature. Western blots were developed using enhanced chemiluminescence reagent. Protein levels were determined by computer-assisted densitometric analysis (GS-800 densitometer, Quantity One; Bio-Rad). The band volumes were determined by standard scanning densitometry with normalization of densitometry measures to β-actin. Each test was performed in triplicate.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Beclin 1 antibody (ab62557) below.

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