All lanes : Anti-Myoferlin antibody [EPR18887] (ab178386) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MYOF knockout HeLa cell lysate
Lane 3 : MDA-MB-231 cell lysate
Lane 4 : SK-BR-3 cell lysate

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: 235 kDa
Observed band size: 180,250 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab178386 observed at 250,180 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab178386 Anti-Myoferlin antibody [EPR18887] was shown to specifically react with Myoferlin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265782 (knockout cell lysate ab257547) was used. Wild-type and Myoferlin knockout samples were subjected to SDS-PAGE. ab178386 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 100% methanol-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Myoferlin with ab178386 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab178386 at 1/100 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Myoferlin with ab178386 at 1/400 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Lanes 1-2 : Anti-Myoferlin antibody [EPR18887] (ab178386) at 1/20000 dilution
Lanes 3-5 : Anti-Myoferlin antibody [EPR18887] (ab178386) at 1/2000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human fetal heart lysate
Lane 5 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 4-5 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 235 kDa
Observed band size: 180,250 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1/5: 10 seconds; Lane 2: 15 seconds; Lane 3/4: 3 minutes.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 22135466, PMID: 23499551).

All lanes : Anti-Myoferlin antibody [EPR18887] (ab178386) at 1/2000 dilution

Lane 1 : Rat kidney lysate
Lane 2 : C6 (rat glial tumor cell line) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : C2C12 (mouse myoblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 235 kDa
Observed band size: 180,250 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1:3 minutes; Lane 2/4: 10 seconds; Lane 3: 3 seconds.

 

Immunofluorescent analysis of 100% methanol-fixed C2C12 (mouse myoblast cell line) cells labeling Myoferlin with ab178386 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on C2C12 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab178386 at 1/100 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.