Anti-MRP1 antibody [EPR22841-78]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MRP1 antibody [EPR22841-78] (AB260038)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293 (human embryonic kidney epithelial cell, Left) / A549 (human lung carcinoma epithelial cell, Right) cells labelling MRP1 with ab260038 at 1/600 (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, ab150077) at 1/2000 dilution 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 (ab260038).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MRP1 antibody [EPR22841-78] (AB260038)

Intracellular Flow Cytometry overlay histogram showing wild-type HeLa (green line) and ABCC1 knockout HeLa cells (ab265256). The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488 pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C.) stained with ab260038 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab260038) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 22°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HeLa - black line ABCC1 knockout HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IP

Unknown

Immunoprecipitation - Anti-MRP1 antibody [EPR22841-78] (AB260038)

MRP1 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate 10ug with ab260038 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab260038 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution. Lane 1 : A549 (human lung carcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab260038 IP in A549 (human lung carcinoma epithelial cell) whole cell lysate, boiled 10ug

Lane 3 : A549 whole cell lysate

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

Exposure time : 3 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260038).

All lanes:

Immunoprecipitation - Anti-MRP1 antibody [EPR22841-78] (ab260038)

Predicted band size: 171 kDa

Observed band size: 190 kDa

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MRP1 antibody [EPR22841-78] (AB260038)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling MRP1 with ab260038 at 1/600 (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, ab150077) at 1/2000 dilution 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 (ab260038).

• IP

Unknown

Immunoprecipitation - Anti-MRP1 antibody [EPR22841-78] (AB260038)

MRP1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10ug with ab260038 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab260038 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution. Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10ug

Lane 2 : ab260038 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate, boiled 10ug

Lane 3 : NIH/3T3 whole cell lysate

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

Exposure time : 75 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260038).

All lanes:

Immunoprecipitation - Anti-MRP1 antibody [EPR22841-78] (ab260038)

Predicted band size: 171 kDa

Observed band size: 190 kDa

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• WB

Lab

Western blot - Anti-MRP1 antibody [EPR22841-78] (AB260038)

Western blot : Rabbit Monoclonal [EPR22841-78] to MRP1 ab260038 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 172 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in abCC1 knockout MCF7 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.

Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MRP1 antibody [EPR22841-78] (ab260038) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

ABCC1 knockout MCF7 at 20 µg

Lane 2:

Western blot - Human ABCC1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-abcc1-knockout-mcf7-cell-line-ab286378'>ab286378</a>) at 20 µg

Lane 3:

DU 145 at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 172 kDa

Observed band size: 172 kDa

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• WB

Lab

Western blot - Anti-MRP1 antibody [EPR22841-78] (AB260038)

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

The expression profile observed is consistent with what has been described in the literature (PMID : 27634913).

Weakly expressing control : K-562, lung, liver, HEK293 (PMID : 18347152, 25880778,11059771, 29615646, 26853103).

Samples (except lane1) are non-boiled as boiling may cause protein aggregates. The Lane 1 lysate was boiled on 100℃ water.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-MRP1 antibody [EPR22841-78] (ab260038) at 1/1000 dilution

Lanes 1 - 2:

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

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 5:

Human lung cancer lysate at 20 µg

Secondary

Lanes 1, 4 and 5:

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

Lanes 2 - 3:

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 1/25000 dilution

Predicted band size: 171 kDa

Observed band size: 120-250 kDa

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• WB

Lab

Western blot - Anti-MRP1 antibody [EPR22841-78] (AB260038)

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

The expression profile observed is consistent with what has been described in the literature (PMID : 27634913).

Weakly expressing control : K-562, lung, liver, HEK293 (PMID : 18347152, 25880778,11059771, 29615646, 26853103).

All lysates were unboiled.

Exposure time : Lanes 1-3 : 10 seconds; Lanes 4-8 : 3 minutes.

All lanes:

Western blot - Anti-MRP1 antibody [EPR22841-78] (ab260038) at 1/1000 dilution

Lane 1:

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

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

Human lung cancer lysate at 20 µg

Lane 5:

Human lung lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 7:

Mouse liver lysate at 10 µg

Lane 8:

Mouse stomach lysate at 10 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

Predicted band size: 171 kDa

Observed band size: 190 kDa

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