All lanes : Anti-MVP antibody [EPR23594-106] (ab273093) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 2 : MVP knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 3 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 99 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab273093 observed at 110 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

ab273093 Anti-MVP antibody [EPR23594-106] was shown to specifically react with MVP in wild-type HeLa cells in Western blot. Significant decrease (8.7 % of intensity compared to the WT band) of signal was observed when MVP knockout cell line ab264817 (knockout cell lysate ab257544) was used.

ab273093 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue labeling MVP with ab273093 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human ovary cancer (PMID: 23739867). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized A549 cells labelling MVP with ab273093 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic staining in A549 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (Human lung carcinoma epithelial cell) cells labelling MVP with ab273093 at 1/50 dilution (1µg) (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). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-MVP antibody [EPR23594-106] (ab273093) at 1/1000 dilution

Lane 1 : Human colon tissue lysate
Lane 2 : Human lung tissue lysate
Lane 3 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 99 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

Exposure time: 10 seconds.

All lanes : Anti-MVP antibody [EPR23594-106] (ab273093) at 1/1000 dilution

Lane 1 : Mouse colon tissue lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 99 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

Exposure time: Lane 1: 26 seconds; Lane 2-4: 70 seconds.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling MVP with ab273093 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

All lanes : Anti-MVP antibody [EPR23594-106] (ab273093) at 1/1000 dilution

Lane 1 : A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : A431 (human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 3 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 5 : HeLa treated with 100U/ml IFN gamma for 24 hours, whole cell lysate
Lane 6 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 99 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 23462207).

MVP expression can be induced by IFN gamma in HeLa cell (PMID: 16418217).

Exposure time: 70 seconds.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling MVP with ab273093 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human lung cancer (PMID: 22117969). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling MVP with ab273093 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in endothelium of human cerebrum (PMID: 14636345). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).