Anti-RAB7 抗体 [EPR7589]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labelling RAB7 with ab137029 at 1 : 250 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1 : 1000 dilution (Green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1 : 200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1 : 1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

ab137029 was shown to react with RAB7 in wild-type HeLa cells in immunocytochemistry with loss of signal observed in RAB7 knockout cell line ab255423. 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 ab137029 at 1/100 dilution 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 µg/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).

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) labelling RAB7 with purified ab137029 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

• ICC/IF

AbReview36175****

Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

ab137029 staining RAB7 in Human HepaRG cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked with 1% milk for 30 minutes at room temperature. Samples were incubated with primary antibody (1/200 in 1% milk) for 30 minutes. An Alexa Fluor®488-conjugated Donkey anti-rabbit IgG polyclonal (1/400) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Alina Macovei

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling RAB7 with ab137029 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma cell line) cells labeling RAB7 with purified ab137029 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling RAB7 with ab137029 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit and anti-rabbit HQ and anti-HQ HRP detection. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab137029 Anti-RAB7 antibody [EPR7589] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-RAB7 knockout cells (red line) stained with ab137029. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab137029, 0.1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG1 isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-RAB7 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min) permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Lanes 1 - 2 : Merged signal (red and green). Green - ab137029 observed at 23 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab137029 was shown to react with RAB7 in wild-type HeLa cells in Western blot with loss of signal observed in RAB7A knockout cell line ab255423 (RAB7A knockout cell lysate ab263831). Wild-type HeLa and RAB7A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab137029 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (ab137029) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAB7A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RAB7A (RAB7) knockout HeLa cell line (<a href='/products/cell-lines/human-rab7a-rab7-knockout-hela-cell-line-ab255423'>ab255423</a>)

Observed band size: 23 kDa

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• WB

Supplier Data

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

ab137029 was shown to react with RAB7 in wild-type HeLa cells in Western blot with loss of signal observed in RAB7 knockout cell line ab255423. Wild-type HeLa and RAB7 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hour before incubation with ab137029 overnight at 4°C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/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-RAB7 antibody [EPR7589] - Late Endosome Marker (ab137029) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 40 µg

Lane 2:

RAB7 knock-out HeLa lysate at 40 µg

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• WB

Unknown

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

All lanes:

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (ab137029) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma cell line) cell lysate at 10 µg

Lane 2:

A431 (human epidermoid carcinoma cell line) cell lysate at 10 µg

Lane 3:

U87 MG (human glioblastoma-astrocytoma epithelial cell line) cell lysate at 10 µg

Lane 4:

HT 1080 (human fibrosarcoma cell line) cell lysate at 10 µg

Lane 5:

L929 (mouse connective tissue fibroblast cell line) cell lysate at 10 µg

Lane 6:

NIH 3T3 (mouse embyro fibroblast cell line) cell lysate at 10 µg

Lane 7:

Raw 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate at 10 µg

Lane 8:

C2C12 (mouse myoblast cell line) cell lysate at 10 µg

Secondary

All lanes:

HRP conjugated Goat anti Rabbit IgG at 1/2000 dilution

false

• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

All lanes:

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (ab137029) at 1/1000 dilution

Lane 1:

A375 (Human malignant melanoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

HT-1080 (Human fibrosarcoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 23 kDa

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• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (AB137029)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : RAB7 knockout HAP1 cell lysate (20 μg)
Lane 3 : A431 cell lysate (20 μg)
Lane 4 : Human fetal brain tissue lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab137029 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab137029 was shown to specifically react with RAB7 in wild-type HAP1 cells. No band was observed when RAB7 knockout samples were examined. Wild-type and RAB7 knockout samples were subjected to SDS-PAGE. ab137029 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (ab137029)

false