Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue sections labeling GAP43 with Purified ab75810 at 1:3000 dilution (0.07 µg/ml). Heat mediated antigen retrieval using Bond© Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used for detection. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-GAP43 antibody [EP890Y] (ab75810) (Purified)

Lane 1 : Human brain lysates
Lane 2 : Mouse brain lysates
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

This antibody fails to detect GAP43 in PC-12 cells which is positve as described in PMID: 21695168 

Immunocytochemistry/ Immunofluorescence analysis of Neuro-2a (Mouse neuroblastoma neuroblast) cells labeling GAP43 with Purified ab75810 at 1:160 dilution (1.4 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab75810 (Purified) at 1:20 dilution (1 µg) immunoprecipitating GAP43 in SH-SY5Y whole cell lysate.
Lane 1 (input): SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10 µg
Lane 2 (+): ab75810 & SH-SY5Y whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab75810 in SH-SY5Y whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling GAP43 with Purified ab75810 at 1:3000 dilution (0.07 µg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used for detection. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling GAP43 with Purified ab75810 at 1:20 dilution (10 µg/ml) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling GAP43 with Purified ab75810 at 1:3000 dilution (0.07 µg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used for detection. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Anti-GAP43 antibody [EP890Y] (ab75810) at 1/50000 dilution (Purified) + SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

ab75810 (unpurified) staining GAP43 in Mouse ear tissue sections by Immunohistochemistry (Formalin/ PFA-fixed paraffin-embedded tissue sections).  The sections were formaldehyde fixed, subjected to heat mediated antigen retrieval at pH 6 and blocked for 10 minutes at 25C. The primary antibody was diluted 1/500 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.

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Overlay histogram showing SH-SY5Y cells stained with ab75810 (unpurified) (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75810, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab75810 (unpurified) stained SKNSH 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 (ab75810, 1/50 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG(H+L) used at a 1/250 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 1.43µM.