Anti-RAB27A + RAB27B antibody [EPR27120-68]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

ab313575 was shown to react with Rab27A in wild-type U-87 MG cells in immunocytochemistry with loss of signal observed in a Rab27A siRNA knockdown cell line. Wild-type and siRNA knockdown 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 ab313575 at 1/500 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 (siRNA knockdown) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and siRNA knockdown 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Raji (human Burkitt's lymphoma B lymphocyte, Left) / SK-MEL-28 (human malignant melanoma cell, Right) cells labelling RAB27A + RAB27B with ab313575 at 1/500 dilution (0.1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. Negative control : Raji (PMID : 15548590).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling RAB27A + RAB27B with ab313575 at 1/5000 (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human skeletal muscle. The section was incubated with ab313575 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling RAB27A + RAB27B with ab313575 at 1/5000 (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human colon (PMID : 26070933). The section was incubated with ab313575 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma tissue labeling RAB27A + RAB27B with ab313575 at 1/5000 (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung adenocarcinoma (PMID : 30480360). The section was incubated with ab313575 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SK-MEL-28 (human malignant melanoma cell) cells labelling RAB27A + RAB27B with ab313575 at 1/100 (5.04 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing cytoplasmic staining in SK-MEL-28. Negative control : Raji (PMID : 15548590). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling RAB27A + RAB27B with ab313575 at 1/100 (5.04 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing both positive staining in 293T cells transfected with human RAB27A and RAB27B expression vectors containing a Myc tag and a Flag tag respectively. Counter stain : ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594), 1 : 100 5ug/ml. Anti-FLAG mouse monoclonal antibody, 1 : 500 2ug/ml. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IP

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Immunoprecipitation - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

RAB27A + RAB27B was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab313575 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab313575 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate Lane 2 : K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab313575 in K562 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/30 dilution

All lanes:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• IP

Lab

Immunoprecipitation - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Immunoprecipitation of Rab27A in U-87 MG cells. Lysates were prepared and immunoprecipitation was performed using 2 µg of ab313575 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

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:

Immunoprecipitation - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 2 µg

Lanes 1 and 3:

U-87 MG cells

Lane 2:

Unbound fraction: Rabbit monoclonal IgG instead of ab313575 in U-87 MG cells

false

• WB

Lab

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

ab313575 was shown to react with Rab27A in wild-type U-87 MG cells in Western blot with loss of signal observed in a Rab27A siRNA knockdown cell line. Cell lysates from wild-type U-87 MG transfected with either scrambled siRNA or Rab27A siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab313575 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-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG transfected with scrambled siRNA control lysate at 20 µg

Lane 2:

U-87 MG transfected with siRNA specifically targeting Rab27A cell lysate at 20 µg

false

• WB

Supplier Data

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : Raji (PMID : 15548590) In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 15 seconds

All lanes:

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

SK-MEL-2 (human skin malignant melanoma cell) whole cell lysate at 20 µg

Lane 3:

Raji (human Burkitts lymphoma B lymphocyte) 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

Observed band size: 27 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : skeletal muscle In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 26 seconds

All lanes:

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

Human stomach tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue 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

Observed band size: 27 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : skeletal muscle. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

Mouse stomach tissue lysate at 20 µg

Lane 2:

Mouse skeletal muscle tissue 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

Observed band size: 27 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : skeletal muscle. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

Rat stomach tissue lysate at 20 µg

Lane 2:

Rat skeletal muscle tissue 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

Observed band size: 27 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (AB313575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody recognizes both RAB27A and RAB27B. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. Exposure time : 3 seconds

All lanes:

Western blot - Anti-RAB27A + RAB27B antibody [EPR27120-68] (ab313575) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containi a His-tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human RAB27A expression vector containi a His-tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human RAB27B expression vector containi a Flag-tag 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

Observed band size: 27 kDa

false

Exposure time: 3s