Anti-Rab5 antibody [EPR21801] - Early Endosome Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and Rab5A knockout Hap1 stained with ab218624 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab218624) (1x 106 in 100μl at 0.2 μg/ml (1/10700)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, Rab5A knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Hap1 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (human breast adenocarcinoma cell line) cell line labelling Rab5 with ab218624 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human cerebrum is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed wild-type and RAB5A KO HAP1 cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing no staining in RAB5A KO HAP1 cell line and granular cytoplasmic staining in parental HAP1 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

ab218624 was shown to react with RAB5A in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a RAB5A knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab218624 overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 ?g/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

These data were 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.

• IP

Supplier Data

Immunoprecipitation - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Rab5 was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma cell line) whole cell lysate with ab218624 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab218624 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : MCF7 whole cell lysate 10 μg (input).

Lane 2 : ab218624 IP in MCF7 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab218624 in MCF7 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

Predicted band size: 24 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in mouse kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in rat liver is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• WB

Lab

Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

ab218624 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 ab218624 overnight at 4 °C at a 1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

These data were 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 [EPR21801] - Early Endosome Marker (ab218624) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

RAB5A Knockout HAP1 lysate at 20 µg

Observed band size: 24 kDa

false

• WB

Supplier Data

Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

ab218624 was shown to specifically react with Rab5 in wild-type HAP1 cells as signal was lost in Rab5 knockout cells. Wild-type and Rab5 knockout samples were subjected to SDS-PAGE. ab218624 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrumentusing the ECL technique.

All lanes:

Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg

Lane 2:

Rab5 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (AB218624)

Exposure time : Lane 1 : 5 seconds; Lanes 2/8 : 15 seconds; Lanes 3/4 : 6 seconds; Lanes 5/7 : 3 seconds; Lane 6 : 26 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624) at 1/1000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma cell line) whole cell lysate at 10 µg

Lane 2:

Human fetal brain lysate at 10 µg

Lane 3:

Human fetal heart lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat heart lysate at 10 µg

Lane 7:

Mouse brain lysate at 10 µg

Lane 8:

Mouse heart lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

false