All lanes : Anti-160 kD Neurofilament Medium antibody [NF-09] - Neuronal Marker (ab7794) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : NEFM knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Observed band size: 150 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab7794 observed at 150 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab7794 Anti-160 kD Neurofilament Medium antibody [NF-09]  was shown to specifically react with 160 kD Neurofilament Medium in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266741 (knockout cell lysate ab257103) was used. Wild-type and 160 kD Neurofilament Medium knockout samples were subjected to SDS-PAGE. ab7794 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) 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^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescence analysis of 1 month neuronal-differentiated dental pulp stem cells, staining 160 kD Neurofilament Medium with ab7794.

Cells were fixed with paraformaldehyde and incubated with primary antibody (1/600). A FITC-conjugated anti-mouse IgG (1/375) was used as the secondary antibody.

All lanes : Anti-160 kD Neurofilament Medium antibody [NF-09] - Neuronal Marker (ab7794) at 1/1000 dilution

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : NEFM knockout HEK-293 whole cell lysate
Lane 3 : A549 whole cell lysate

Lysates/proteins at 20 µg per lane.

Observed band size: 150 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab7794 observed at 150 kDa. Red - loading control, ab52866, observed at 50 kDa.

ab7794 was shown to specifically react with NEFM (Neurofilament) in wild-type HEK-293 cells as signal was lost in NEFM knockout cells. Wild-type and NEFM knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab7794 and ab52866 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

 

Western blotting analysis of neurofilament medium protein in porcine brain lysate (reducing conditions) by mouse monoclonal NF-09.

ab7794 staining Neurofilament medium protein in mouse Neuro2A cells by ICC/IF.

ICC/IF image of ab7794 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7794, 1æg/ml) overnight at +4øC. The secondary antibody (green)ÿwas Alexa Fluor© 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor© 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43æM.