Recombinant Anti-Pan Trk antibody [EP1058Y] (ab76291)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of (A) His-human TrkA overexpression 293T whole cell pellet, (B) His-human TrkB overexpression 293T whole cell pellet, (C) His-human TrkC overexpression 293T whole cell pellet and (D) HEK-293T transfected with empty plasmid labelling Pan Trk with purified ab76291 at 1/1000. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. 

Positive staining on (A) His-human TrkA overexpression 293T whole cell pellet, (B) His-human TrkB overexpression 293T whole cell pellet and (C) His-human TrkC overexpression 293T whole cell pellet.
No staining on (D) HEK-293T transfected with empty plasmid.
The section was incubated with abab76291 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

This antibody has relatively lower affinity to TrkC compared to TrkA and TrkB.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on mouse cerebrum.
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunocytochemistry/immunofluorescence analysis of SH-SY5Y (Human neuroblastoma epithelial cell) labeling pan Trk with ab76291 at 1/100 dilution (7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/mL).

Confocal image showing cytoplasmic staining in SH-SY5Y cell line.

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Pan Trk with ab76291 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Intracellular Flow Cytometry analysis of293T (human embryonic kidney epithelial cell) transfected with TrkB overexpression construct labeling Pan Trk with ab76291 at 1/700 dilution (0.1 µg) (Right). The cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody.Isotype control/colour: Rabbit monoclonal IgG (ab172730) (Left). 

Intracellular Flow Cytometry analysis of293T (human embryonic kidney epithelial cell) transfected with TrkA overexpression construct labeling Pan Trk with ab76291 at 1/700 dilution (0.1 µg) (Right). The cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.Isotype control/colour: Rabbit monoclonal IgG (ab172730) (Left). 

Immunocytochemistry/immunofluorescence analysis of TrkA- overexpressed 293T (human embryonic kidney epithelial cell) labelled with the pan Trk antibody ab76291 at 1/1000 dilution (0.7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkA expression vector.

Immunocytochemistry/immunofluorescence analysis of TrkB- overexpressed 293T (human embryonic kidney epithelial cell) labelled with the pan Trk antibody ab76291 at 1/1000 dilution (0.7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkB expression vector.

Immunocytochemistry/immunofluorescence analysis of TrkC- overexpressed 293T (human embryonic kidney epithelial cell) labelled with the pan Trk antibody ab76291 at 1/1000 dilution (0.7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkC expression vector.

ab76291 at 1/40 immunoprecipitating Pan Trk in human brain tissue lysate observed at 145 kDa.

Lane 1 (input): Human brain tissue lysate (10µg)
Lane 2 (+): ab76291+ human brain tissue lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76291 in human brain lysate
For western blotting, ab76291 at 1/1000 dilution (0.7 μg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 were used.

The 30 kDa band is an intracellular fragment, and the 140 kDa observed MW which is higher than the predicted one is due to the glycosylation modification. (refer to ab189903).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

ab76291 at 1/40 immunoprecipitating Pan Trk in mouse brain tissue lysate observed at 145 kDa.

Lane 1 (input): Mouse brain tissue lysate (10µg)
Lane 2 (+): ab76291+ mouse brain tissue lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76291 in Mouse brain lysate
For western blotting, ab76291 at 1/1000 dilution (0.7 μg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 were used.

The 30 kDa band is an intracellular fragment, and the 140 kDa observed MW which is higher than the predicted one is due to the glycosylation modification. (refer to ab189903).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

Overlay histogram showing SH-SY5Y (human neuroblastoma cell line from bone marrow) cells stained with unpurified ab76291 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76291, 1/50 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 monoclonal IgG (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 methanol (5 min) used under the same conditions.Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.  Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on human cerebrum.
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on rat cerebrum.
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Negative control: No staining on human liver.

The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Unpurified ab76291 staining Pan Trk in murine brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
Tissue was fixed with formaldehyde, permeabilized with 0.1% Saponin/PBS and blocked with 4% serum for 30 minutes at 25°C, antigen retrieval was by heat mediation with a citrate buffer. Samples were incubated with primary antibody (1/150 in blocking buffer) for 16 hours at 4°C. A FITC-conjugated goat anti-rabbit polyclonal IgG (1/100) was used as the secondary antibody.

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ICC/IF image of Pan Trk staining on culture of mouse DRG neurons using unpurified ab76291 (1/100). The cells were fixed using formaldehyde and permeabilized using 0.2% Triton X-100. The cells were blocked using 10% Goat serum for 1 hour at 22°C. Unpurified ab76291 was diluted 1/100 using PBS and incubated with the cells for 30 mins at 22°C. The secondary antibody used was Goat polyclonal to Rabbit IgG conjugated to Alexa Fluor^® 488 (1/1000). Neuron was stained using Beta III tubulin antibody (Alexa Fluor^® 647)

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