重组Anti-RAB7抗体[EPR7588(B)] - Late Endosome Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

Immunohistochemical analysis of paraffin-embedded Human colon adenocarcinoma tissue labelling Rab7A with unpurified ab126712 at dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

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Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

All lanes:

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (ab126712) at 1/1000 dilution

Lane 1:

A375 cell lysate at 10 µg

Lane 2:

A431 cell lysate at 10 µg

Lane 3:

HACAT cell lysate at 10 µg

Lane 4:

Human fetal brain lysate at 10 µg

Lane 5:

B16F0 cell lysate at 10 µg

Lane 6:

C6 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

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• WB

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Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (ab126712) at 1/1000 dilution

Lane 1:

A375 whole cell lysate at 20 µg

Lane 2:

A431 whole cell lysate at 20 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

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Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (ab126712) at 1/1000 dilution

Lane 1:

C6 (rat glioma) whole cell lysate at 20 µg

Lane 2:

Raw264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryo) whole cell lysate at 20 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

Predicted band size: 35 kDa,36 kDa

Observed band size: 23 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

Immunohistochemical staining of paraffin-embedded human bladder carcinoma sections labelling RAB7 with purified ab126712 at dilution of 1/70. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• WB

Lab

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

Lanes 1 - 2 : Merged signal (red and green). Green - ab126712 observed at 23 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab126712 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 ab126712 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 [EPR7588(B)] - Late Endosome Marker (ab126712) 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

Lab

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

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 - ab126712 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab126712 was shown to recognize RAB7 when RAB7 knockout samples were used, along with additional cross-reactive bands. Wild-type and RAB7 knockout samples were subjected to SDS-PAGE. ab126712 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 and incubated overnight at 4°C. Blots were developed withGoat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (ab126712)

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• OI-RD Scanning

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OI-RD Scanning - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker (AB126712)

RAB7 western blot using anti-RAB7 antibody [EPR7588(B)] - Late Endosome Marker ab126712. Publication image and figure legend from Li, M., Zhang, D., et al., 2020, Front Microbiol, PubMed 32210929.

ab126712 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab126712 please see the product overview.

Effects of Rab5 and Rab7 knockdown on Zika virus (ZIKV) infection. (A) The knockdown efficiency of Rab5 or Rab7 was determined by Western blot. T98G cells were transfected with either siControl, siRab5, or siRab7 for 48 h. Cell lysates were reacted with an anti-Rab5 antibody or an anti-Rab7 antibody specific for the indicated proteins. β-actin was used as an internal loading control. One representative experiment out of three is shown. (B) Rab5 or Rab7 depletion reduced ZIKV propagation. T98G cells transfected with the indicated siRNAs were infected with ZIKV at a multiplicity of infection (MOI) of 0.5 and incubated for 48 h to allow virus propagation. The infected cells were lysed to quantitate viral RNA copy numbers by RT-qPCR. (C) At 48 h post-infection, the titers of supernatant viruses were determined by plaque assay. (D) ZIKV localized in Rab5- and Rab7-postitive endosomes. T98G cells were transfected with EGFP-tagged Rab5 or Rab7 for 24 h, followed by infection with ZIKV at an MOI of 10 at 4°C for 1 h and then shifted to 37°C. At 24 h post-infection, the cells were fixed and stained with an anti-ZIKV envelop antibody (red). Nuclei were stained with DAPI (blue). One representative experiment out of three is shown (A). The immunofluorescence was examined under a confocal microscope. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm (D). The data shown are the mean ± SD of three independent experiments (B,C). *p < 0.05; **p < 0.01; ***p < 0.001.

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