WB
Western blot analysis of ATG5 on different lysates with Rabbit anti-ATG5 antibody at 1/5,000 dilution. Lane 1: HeLa cell lysate (20 µg/Lane), Lane 2: A431 cell lysate (20 µg/Lane), Lane 3: PC-12 cell lysate (20 µg/Lane), Lane 4: NIH/3T3 cell lysate (20 µg/Lane), Lane 5: Neuro-2a cell lysate (20 µg/Lane), Exposure time: 1 minute; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/100,000 dilution was used for 1 hour at room temperature.IHC
Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-ATG5 antibody at 1/2,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells labeling ATG5 with Rabbit anti-ATG5 antibody at 1/250 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-ATG5 antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.FC
Flow cytometric analysis of NIH/3T3 cells labeling ATG5. Cells were fixed and permeabilized. Then stained with the primary antibody (1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).IP
ATG5 was immunoprecipitated in 0.2mg HeLa cell lysate with Rabbit anti-ATG5 antibody at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using Rabbit anti-ATG5 antibody at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input), Lane 2: Rabbit IgG instead of ERabbit anti-ATG5 antibody in HeLa cell lysate, Lane 3: Rabbit anti-ATG5 antibody IP in HeLa cell lysate. Blocking/Dilution buffer: 5% NFDM/TBST, Exposure time: 1 minute 21 seconds.| Product Name | ATG5 Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Product description | Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer. The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles. This conjugation reaction is mediated by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10. |
| Immunogen | Synthetic peptide within Human ATG5 aa 1-50 / 275. |
| Clonality | Monoclonal |
|---|---|
| Isotype | IgG |
| Host Species | Recombinant rabbit |
| Tested Applications | FCICC/IFIHCIPWB |
| WB:1:5000-1:10000 IHC:1:50-1:200 ICC/IF:1:100-1:250 FC:1:1000 IP:1-2μg/sample |
|
| Species Reactivity | HumanMonkeyMouseRat |
| Concentration | 1mg/ml |
| Purification | Protein A |
| Alternative Names | APG 5 antibody APG 5L antibody APG5 antibody APG5 autophagy 5 like antibody APG5 like antibody APG5-like antibody APG5L antibody Apoptosis specific protein antibody Apoptosis-specific protein antibody ASP antibody ATG 5 antibody Atg5 antibody ATG5 autophagy related 5 homolog antibody ATG5_HUMAN antibody Autophagy protein 5 antibody Autophagy related 5 antibody hAPG5 antibody Homolog of S Cerevisiae autophagy 5 antibody OTTHUMP00000040507 antibody |
|---|---|
| Molecular Weight(MW) | 32 kDa(Observed band size: 55 kDa) |
| Cellular Localization | Cytoplasm, Preautophagosomal structure membrane. |
WB
Western blot analysis of ATG5 on different lysates with Rabbit anti-ATG5 antibody at 1/5,000 dilution. Lane 1: HeLa cell lysate (20 µg/Lane), Lane 2: A431 cell lysate (20 µg/Lane), Lane 3: PC-12 cell lysate (20 µg/Lane), Lane 4: NIH/3T3 cell lysate (20 µg/Lane), Lane 5: Neuro-2a cell lysate (20 µg/Lane), Exposure time: 1 minute; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/100,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-ATG5 antibody at 1/2,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells labeling ATG5 with Rabbit anti-ATG5 antibody at 1/250 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-ATG5 antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
FC
Flow cytometric analysis of NIH/3T3 cells labeling ATG5. Cells were fixed and permeabilized. Then stained with the primary antibody (1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
IP
ATG5 was immunoprecipitated in 0.2mg HeLa cell lysate with Rabbit anti-ATG5 antibody at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using Rabbit anti-ATG5 antibody at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input), Lane 2: Rabbit IgG instead of ERabbit anti-ATG5 antibody in HeLa cell lysate, Lane 3: Rabbit anti-ATG5 antibody IP in HeLa cell lysate. Blocking/Dilution buffer: 5% NFDM/TBST, Exposure time: 1 minute 21 seconds.| Application Notes | WB:1:5000-1:10000 IHC:1:50-1:200 ICC/IF:1:100-1:250 FC:1:1000 IP:1-2μg/sample |
|---|
| Form | Liquid |
|---|---|
| Storage Instructions | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage Buffer | 1*TBS (pH7.4), 1%BSA, 40%Glycerol. Preservative: 0.05% Sodium Azide. |
