Non-fixed and 4% formaldehyde-fixed C6 (rat glial tumor cell line) cells labeling p75 NGF Receptor using ab245134 at 2 µg/ml for 1 hour (green) in ICC/IF.

Blocking: 10% normal horse serum, 30 mins.

Secondary antibody: Donkey anti-mouse-CF488A at 2 µg/mL for 1 hour.

Nuclei stained with Hoechst (blue).

Flow Cytometric analysis of SH-SY5Y (human neuroblastoma cell line from bone marrow) (Left panel) and C6 (rat glial tumor cell line) (Right panel) staining p75 NGF Receptor using ab245134 (red lines) and a different antibody (solid black lines) at 2 µg per 10⁶ cells, 60 mins on ice. Negative control is the dashed line, Non specific Control IgG.

Blocking: 200 µg/mL sheep IgG, 30 minutes on ice.

Secondary antibody: Goat anti-mouse-PE used at a 1/100 dilution for 20 mins in the dark on ice.

Immunoprecipitation - Anti-p75 NGF Receptor antibody [8J2] (ab245134) (10 µg antibody per 200 µg total protein):

Lane 1: p75 NGF Receptor IP in C6 (rat glial tumor cell line) whole cell lysate.

Lane 2: Control Protein G agarose (10 µL).

Precipitated p75 NGF Receptor was eluted off the beads and antibody by heating and addition of SDS-PAGE sample buffer.

WB method: ab245134 (1 µg/mL) and secondary anti-sheep-HRP (1/6000) antibody.

Detection: Chemiluminiscence.

 

All lanes : Anti-p75 NGF Receptor antibody [8J2] (ab245134) at 1 µg/ml (overnight at 4°C)

Lane 1 : Mouse p75 NGF Receptor-transfected cells
Lane 2 : Non-transfected control cells
Lane 3 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate
Lane 4 : C6 (rat glial tumor cell line) cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : anti-mouse-HRP, 1 hour at RT at 1/6000 dilution

Performed under non-reducing conditions.

Predicted band size: 45 kDa

SDS-PAGE: 4-12%, non-reducing conditions. 

Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.45 µm).

Detection: Chemiluminiscence.