Anti-RAB7 antibody [EPR7589] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137029).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137029).

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] - BSA and Azide free (AB214806)

Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137029).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137029).

• ICC/IF

AbReview36175****

Immunocytochemistry/ Immunofluorescence - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137029).

This image is courtesy of an Abreview submitted by Alina Macovei

• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

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] - BSA and Azide free (ab214806) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAB7A knockout HeLa cell lysate at 20 µg

Observed band size: 23 kDa

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• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7589] - BSA and Azide free (AB214806)

This data was developed using ab137029, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

All lanes:

Western blot - Anti-RAB7 antibody [EPR7589] - Late Endosome Marker (<a href='/en-us/products/primary-antibodies/rab7-antibody-epr7589-late-endosome-marker-ab137029'>ab137029</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

RAB7 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Wild-type HeLa whole cell lysate at 20 µg

Lane 4:

RAB7A knockout HeLa whole cell lysate at 20 µg

Lane 5:

A375 (human malignant melanoma cell line) cell lysate at 20 µg

Lane 6:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Lane 7:

Raw 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) 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 1/20000 dilution

Observed band size: 23 kDa

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Exposure time: 20s