Anti-LIMPII antibody [EPR26243-125]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SCARB2 KO MCF7 (SCARB2 knockout human breast adenocarcinoma epithelial cell) cells labelling LIMPII with ab314217 at 1/50 (10.54 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing no staining in SCARB2 KO MCF7 cells, and lysosomal staining in parental MCF7 cells.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab25630 Anti-LAMP1 mouse monoclonal antibody was used to counterstain tubulin at 1/200 0.2ug/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 2ug/ml dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LIMPII antibody [EPR26243-125] (AB314217)

ab314217 was shown to react with SCARB2 in wild-type MCF7 cells in immunocytochemistry with loss of signal observed in SCARB2 knockout cell line ab274952. 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 ab314217 at 1/50 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SCARB2 KO MCF7 (human SCARB2 knockout breast adenocarcinoma epithelial cell, Left) / Parental MCF7 (Right) cells labelling LIMPII with ab314217 at 1/50 dilution (1 ug)/Red (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling LIMPII with ab314217 at 1/2000 (0.264 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Punctate staining on human cerebrum.The section was incubated with ab314217 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-LIMPII antibody [EPR26243-125] (AB314217)

Immunohistochemical analysis of paraffin-embedded Human kindey tissue labeling LIMPII with ab314217 at 1/2000 (0.264 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Punctate staining on human kidney.The section was incubated with ab314217 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-LIMPII antibody [EPR26243-125] (AB314217)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling LIMPII with ab314217 at 1/2000 (0.264 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Punctate staining on human liver.The section was incubated with ab314217 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-LIMPII antibody [EPR26243-125] (AB314217)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type MCF7 (human breast adenocarcinoma epithelial cell) cell pellet (B) SCARB2 knockout MCF7 cell pellet tissue labeling LIMPII with ab314217 at 1/100 (5.27 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Punctate staining on (A) MCF7 cell pellet, no staining on (B) SCARB2 knockout MCF7 cell pellet.The section was incubated with ab314217 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

• IP

Collaborator

Immunoprecipitation - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Immunoprecipitation of SCARB2 in MCF7 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab314217 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.

false

• IP

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Immunoprecipitation - Anti-LIMPII antibody [EPR26243-125] (AB314217)

LIMPII was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate with ab314217 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314217 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate Lane 2 : ab314217 IP in MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314217 in MCF7 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-LIMPII antibody [EPR26243-125] (ab314217) at 1/30 dilution

All lanes:

MCF7 (human breast adenocarcinoma epithelial cell) 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: 8s

• WB

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Western blot - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-LIMPII antibody [EPR26243-125] (ab314217) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

293T (human embryonic kidney epithelial cell) 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: 75 kDa

false

Exposure time: 15s

• WB

Collaborator

Western blot - Anti-LIMPII antibody [EPR26243-125] (AB314217)

ab314217 was shown to react with SCARB2 in wild-type MCF7 cells in Western blot with loss of signal observed in SCARB2 knockout cell line ab274952. Wild-type MCF7 and SCARB2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab314217 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-LIMPII antibody [EPR26243-125] (ab314217) at 1/1000 dilution

Lane 1:

Wild-type MCF7 lysate at 30 µg

Lane 2:

SCARB2 knock-out MCF7 lysate at 30 µg

Lane 2:

Western blot - Human SCARB2 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-scarb2-knockout-mcf7-cell-line-ab274952'>ab274952</a>) at 30 µg

false

• WB

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Western blot - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, ab314217 was shown to bind specifically to LIMPII. A band was observed at 75 kDa in wild-type MCF7 cell lysates whereas loss of signal was observed in the SCARB2 knockout cell line ab274952(knockout cell lysate ab275010). In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-LIMPII antibody [EPR26243-125] (ab314217) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SCARB2 knockout MCF7 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) 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: 75 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-LIMPII antibody [EPR26243-125] (AB314217)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-LIMPII antibody [EPR26243-125] (ab314217) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human liver 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: 75 kDa

false

Exposure time: 15s