Anti-Neurofibromin antibody [EPR22989-68]

• WB

Lab

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (AB238142)

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:

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

NFIB knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human NF1 (Neurofibromin) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-nf1-neurofibromin-knockout-hela-cell-line-ab264725'>ab264725</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 319 kDa

Observed band size: 319 kDa

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• WB

Lab

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (AB238142)

Anti-NF1 antibody [EPR22989-68] (ab238142) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab238142 was shown to bind specifically to NF1. A band was observed at 180-270 kDa in wild-type MCF7 cell lysates with no signal observed at this size in NF1 knockout cell line. To generate this image, wild-type and NF1 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lanes 1 - 4:

Western blot at 20 µg

Lane 2:

Western blot - Human NF1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-nf1-knockout-mcf7-cell-line-ab286416'>ab286416</a>) at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 319 kDa

false

• WB

Lab

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (AB238142)

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:

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Neurofibromin knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 319 kDa

Observed band size: 250 kDa,300 kDa

false

• WB

Lab

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (AB238142)

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.

All lanes:

Western blot - Anti-Neurofibromin antibody [EPR22989-68] (ab238142) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg

Lane 5:

PC-12 (rat adrenal gland heochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 319 kDa

Observed band size: 200 kDa,250 kDa,300 kDa

false