Anti-GAP43 抗体 [EP890Y] - Neuronal Marker

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

Intracellular 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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling GAP43 with ab75810 at 1ug/ml followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining in human Alzheimer's brain.

The section was incubated with ab75810 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebrum labelling GAP43 with ab75810 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32 mins. ab75810 anti-GAP43 antibody [EP890Y] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

• IP

Lab

Immunoprecipitation - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75810)

All lanes:

Immunoprecipitation - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (ab75810)

Predicted band size: 24 kDa,45 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

This image is a courtesy of anonymous Abreview.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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.

• WB

Lab

Western blot - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

All lanes:

Western blot - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (ab75810) at 1/50000 dilution

All lanes:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 24 kDa

Observed band size: 48 kDa

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• WB

Lab

Western blot - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (AB75810)

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

All lanes:

Western blot - Anti-GAP43 antibody [EP890Y] - Neuronal Marker (ab75810)

Lane 1:

Human brain lysates at 20 µg

Lane 2:

Mouse brain lysates at 20 µg

Lane 4:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 24 kDa

Observed band size: 48 kDa

false