All lanes : 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 & 4-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/25000 dilution

Predicted band size: 171 kDa
Observed band size: 120-250 kDa why is the actual band size different from the predicted?

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.

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling MRP1 with ab260038 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and weakly cytoplasmic staining on mouse stomach (PMID: 23667609). The section was incubated with ab260038 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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.

All lanes : 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 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 171 kDa
Observed band size: 190 kDa why is the actual band size different from the predicted?

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.

MRP1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 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

Lane 4: Rabbit monoclonal IgG (ab172730) instead of ab260038 in NIH/3T3 whole cell lysate.

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

Exposure time: 75 seconds

 

MRP1 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate  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

Lane 4: Rabbit monoclonal IgG (ab172730) instead of ab260038 in A549 whole cell lysate

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

Exposure time: 3 seconds.

 

Immunohistochemical analysis of paraffin-embedded human lung carcinoma and the paired adjacent lung tissue tissue labeling MRP1 with ab260038 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and weakly cytoplasmic staining on human lung carcinoma (panel A) and no staining on the paired adjacent lung tissue (panel B, PMID: 25880778). The section was incubated with ab260038 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human gastric adenocarcinoma tissue labeling MRP1 with ab260038 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and weakly cytoplasmic staining on human gastric adenocarcinoma. The section was incubated with ab260038 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling MRP1 with ab260038 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and weakly cytoplasmic staining on human breast carcinoma (PMID: 15517899). The section was incubated with ab260038 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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.

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.

Negative control: HEK-293 (PMID: 29615646，PMID:12235150).