Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

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.

• WB

Supplier Data

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

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.

All lanes:

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (ab288769) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Rab5 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 24 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

ab288769 was shown to react with RAB5A in wild-type HAP1 cells in Western blot with loss of signal observed in a RAB5A knockout cell line. Wild-type HAP1 and RAB5A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab288769 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (ab288769) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

RAB5A knock-out HAP1 lysate at 20 µg

false

• WB

Supplier Data

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (AB288769)

Blocking and diluting buffer : 5% NFDM/TBST

Exposure time : 70 seconds

All lanes:

Western blot - Anti-Rab5 antibody [1/Rab5] - Early Endosome Marker (ab288769) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Human heart tissue lysate at 20 µg

Lane 3:

Human kidney tissue lysate at 20 µg

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

false