All lanes : Anti-NGF antibody [EP1320Y] (ab52918) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NGF knockout HeLa cell lysate
Lane 3 : Human fetal brain tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 27 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab52918).

Lanes 1-3: Merged signal (red and green). Green - ab52918 observed at 32 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab52918 Anti-NGF antibody [EP1320Y] was shown to specifically react with NGF in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264770 (knockout cell lysate ab257004) was used. Wild-type and NGF knockout samples were subjected to SDS-PAGE. ab52918 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 10000 dilution for 1 hour at room temperature before imaging.

 

 

Immunofluorescence staining of U87-MG cells with purified ab52918 at a working dilution of 1 in 300, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab52918 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52918).

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab52918 at a working dilution of 1 in 250. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52918).

Immunohistochemical staining of paraffin embedded human brain using unpurified b52918 at 1/50-1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52918).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical staining of NGF mouse tissue sections (formalin/ PFA-fixed paraffin-embedded tissue sections) with unpurified ab52918. The sections were formaldehyde fixed, subjected to heat mediated antigen retrieval and blocked for 10 minutes at 25°C. The primary antibody was diluted 1/50 and incubated with the sample for 1 hour at 25°C. An HRP polymer anti-rabbit IgG system was used undiluted, as the secondary antibody.

 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52918).