All lanes : Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MTAP knockout HeLa cell lysate
Lane 3 : HT-29 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab254265 observed at 32 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab254265 Anti-MTAP antibody [EPR22570-76] was shown to specifically react with MTAP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265272 (knockout cell lysate ab257194) was used. Wild-type and MTAP knockout samples were subjected to SDS-PAGE. ab254265 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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.

 

All lanes : Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1 : Mouse skin lysate
Lane 2 : Mouse liver lysate
Lane 3 : Rat skin lysate
Lane 4 : Rat liver lysate
Lane 5 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times:
Lanes 1-4: 15 seconds; Lane 5: 10 seconds; Lane 6: 3.25 seconds.

The expression molecular weight observed is consistent with what has been described in the literature (PMID:15492751).

 

All lanes : Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1 : Human liver lysate
Lane 2 : Human placenta lysate
Lane 3 : HT-29 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : HeLa (human epithelial cell line from cervix adenocarcinoma0 whole cell lysate
Lane 6 : Raji 9human Burkitt's lymphoma cell line) whole cell lysate
Lane 7 : Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 8 : A549 9human colorectal adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 3-8 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Negative control: A549 (PMID: 17548352).

The expression molecular weight observed is consistent with what has been described in the literature (PMID:15492751).

Exposure times.

Lanes 1-2: 10 seconds; Lane3: 5.5 seconds; Lanes 4-8: 3.25 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) (Left) anf H-29 (human colorectal adenocarcinoma cell line) (Right) cells labeling MTAP with ab254265 at 1/400 dilution (red) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control: A549（PMID: 8971171.

 

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in rat liver (PMID:15492751) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

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

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in mouse liver (PMID:15492751) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

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

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in stroma cells and no staining in tumor cells of human bladder cancer (PMID:27270441) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

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

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in human breast (PMID:26751376, 18712977) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) and HT-29 (human colorectal adenocarcinoma cell line) cells labeling MTAP with ab254265 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining in HT-29 cell line. The nuclear counter stain is DAPI (blue) Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

SEcondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Negative control: A549（PMID: 8971171.