Recombinant Anti-Rab5 antibody [1/Rab5] (ab288769)

All lanes : Anti-Rab5 antibody [1/Rab5] (ab288769) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate
Lane 2 : Rab5 knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution

Predicted band size: 24 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Performed under reducing conditions.

False colour image of Western blot: Anti-Rab5 antibody (ab288769) staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab288769 was shown to bind specifically to Rab5. A band was observed at 24 kDa in wild-type HAP1 cell lysates with no signal observed at this size in Rab5 knockout cell lysates. To generate this image, wild-type and Rab5 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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.

Immunohistochemical analysis of paraffin-embedded human astrocytoma tissue labeling Rab5 with ab288769 at 1/100 (8.35 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human astrocytoma.

The section was incubated with ab288769 for 30 mins at room temperature and followed by specific mouse IgG2a antibody for 8mins. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-Rab5 antibody [1/Rab5] (ab288769) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Human heart tissue lysate
Lane 3 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 24 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 70 seconds

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Rab5 with ab288769 at 1/100 (8.35 μg/ml) dilution followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human breast carcinoma. The section was incubated with ab288769 for 30 mins at room temperature and followed by specific mouse IgG2a antibody for 8mins. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. The sample was counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Rab5 with ab288769 at 1/100 (8.35 μg/ml) dilution followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human placenta. The section was incubated with ab288769 for 30 mins at room temperature and followed by specific mouse IgG2a antibody for 8mins. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. The sample was counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Rab5 with ab288769 at 1/100 (8.35 μg/ml) dilution followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human kidney. The section was incubated with ab288769 for 30 mins at room temperature and followed by specific mouse IgG2a antibody for 8mins. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. The sample was counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.