重组Anti-RAB7抗体[EPR7589] (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.

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.

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.

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).

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. 

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.

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. 

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.

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