All lanes : Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NFIB knockout HeLa cell lysate
Lane 3 : HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 319 kDa

Lanes 1-3: Merged signal (red and green). Green - ab238142 observed at 319 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab238142 Recombinant Anti-NF1 antibody [EPR22989-68] was shown to specifically react with NFIB in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264725 (knockout cell lysate ab258533) was used. Wild-type and NF1 knockout samples were subjected to SDS-PAGE. ab238142 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.

 

All lanes : Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Neurofibromin knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 319 kDa
Observed band size: 26 seconds kDa why is the actual band size different from the predicted?

ab238142 was shown to specifically react with Neurofibromin in wild-type HAP1 cells as signal was lost in Neurofibromin knockout cells. Wild-type and Neurofibromin knockout samples were subjected to SDS-PAGE. ab238142 and ab129002 (Rabbit anti-Vinculin loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/5000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique. Lysate should be made freshly and used in WB immediately to minimize protein degradation. Degraded fragment(250 KD) observed is consistent with what has been described in the literature (PMID: 30131853, PMID: 30408279).

 

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

Exposure time: 26 seconds

All lanes : Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland heochromocytoma) 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: 319 kDa
Observed band size: 200,250,300 kDa why is the actual band size different from the predicted?

Degraded fragments (200-250 KD) observed is consistent with what has been described in the literature (PMID: 30131853, PMID: 30408279). 

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

Exposure time: Lane 1:3 minutes; Lane 2:26 seconds; Lanes 3-5: 3 minutes.