An autophagosome is a spherical structure with double layer membranes. It is a central organelle in autophagy, the process of degrading bulk cytoplasmic contents including damaged organelles. The autophagosome encloses the materials to be degraded and then fuses with the lysosome which contains hydrolases and other degradation enzymes.
Autophagy occurs regularly in the cell but increases in circumstances of hormonal stimulation, starvation, and drug treatments. It is crucial in such cellular processes as cell differentiation, aging, and cell death. Defects in the process of autophagy are linked to several human diseases including muscular disorders, cancer, neurodegenerative diseases, and pathogen infections.
Autophagosome marker antibodies can aid in the study of autophagosome structure, function, and formation Invitrogen autophagosome marker antibodies are designed to dependably detect the key autophagosome targets. Each antibody is validated for use in various applications. Key autophagosome marker targets include:
Immunofluorescence analysis of Rab11 on 70% confluent log-phase U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Rab11 rabbit polyclonal antibody (Cat. No. 71-5300) at 2 µg/mL in 1% BSA, incubated for 3 hours at room temperature, and then labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Cat. No. A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade™ Gold Antifade Mountant DAPI (Cat. No. S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594-conjugated phalloidin (Cat. No. A12381). Panel d is a merged image showing cytoplasmic localization. Panel e is a negative control (no primary antibody). The images were captured at 20x magnification.
Chromatin immunoprecipitation analysis of MAP1LC3B was performed using cross-linked chromatin from 1 x 106 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay with 1.0 µL/100 µL well volume of a MAP1LC3B rabbit monoclonal antibody (Cat. No. 700712). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify the promoter region of human UBE2B, or the imprinting control region (ICR) of the human H19 locus. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the human UBE2B and H19 loci are shown above the data where boxes represent exons (grey boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by UBE2B and H19 primers are represented by black bars.