Recombinant Anti-APG5L/ATG5 antibody [EPR1755(2)] - Mouse IgG1 (Chimeric) (ab307842)

Blocking/Dilution buffer: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

False colour image of Western blot: Anti-APG5L/ATG5 antibody [EEPR1755(2)] (ab307842) staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody [16891] (ab181602) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab307842 was shown to bind specifically to APG5L/ATG5 . A band was observed at 55 kDa in wild-type HAP1 cell lysates with no signal observed at this size in APG5L/ATG5 knockout cell line. To generate this image, wild-type and APG5L/ATG5 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution.

Exposure time: Lanes 1-3: 48 seconds;Lanes 4-9: 92 seconds;Lanes 10-13: 37 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling APG5L/ATG5 with ab307842 at 1/500 (1.66 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Cytoplasmic staining on rat colon.

The section was incubated with ab307842 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling APG5L/ATG5 with ab307842 at 1/500 (1.66 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Cytoplasmic staining on mouse colon.

The section was incubated with ab307842 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded cell pellets labeling APG5L/ATG5 with ab307842 at 1/2000 dilution (0.415 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Cytoplasmic staining on (A) parental HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet, no staining on (B) APG5L/ATG5 knockout HAP1 (HO010083) cell pellet.

The section was incubated with ab307842 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling APG5L/ATG5 with ab307842 at 1/500 (1.66 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Cytoplasmic staining on human ovarian cancer.

The section was incubated with ab307842 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.