Flow Cytometry - PSD95 antibody [6G6-1C9] (ab2723)

Overlay histogram showing SH-SY5Y cells stained with ab2723 (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 (ab2723, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Western blot - PSD95 antibody [6G6-1C9] (ab2723)

All lanes : Anti-PSD95 antibody [6G6-1C9] (ab2723) at 5 µg/ml

Lane 1 : Hippocampus (Rat) Tissue Lysate
Lane 2 : Forebrain (Rat) Tissue Lysate
Lane 3 : Hippocampus (Mouse) Tissue Lysate
Lane 4 : Forebrain (Mouse) Tissue Lysate

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 80 kDa
Observed band size : 95 kDa (why is the actual band size different from the predicted?)
Additional bands at : 100 kDa,110 kDa,80 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 3 minutes

Immunocytochemistry/ Immunofluorescence - PSD95 antibody [6G6-1C9] (ab2723)

ab2723 at 1/200 staining rat primary hippocampal neurons by ICC/IF. The cells were paraformaldehyde fixed and then permeabilized with 0.1% Triton X-100 before being stained with the antibody for 1 hour. An Alexa-Fluor ® 594 conjugated goat polyclonal antibody was used as the secondary.

The antibody shows numerous puncta along the shafts.

This image is courtesy of an Abreview submitted by Dr Farida Korobova

Western blot - PSD95 antibody [6G6-1C9] (ab2723)

Predicted band size : 80 kDa


All lanes : PSD95 antibody (ab2723) at 1/2000 dilution (in blocking buffer for 16 hours at 4°C) + whole tissue lysate of Mouse hippocampus at 10µg

Secondary
An HRP-conjugated Horse anti-mouse IgG polyclonal at 1/10000 dilution
developed using the ECL technique

Performed under reducing conditions

Exposure time : 30 seconds

Blocking Step : 5% Milk for 1 hour at 25°C

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Anti-PSD95 antibody [6G6-1C9] (ab2723)

ab2723 staining cultured rat primary hippocampal neurons by ICC/IF. The cultured neurons were fixed with 4% formaldehyde for 5 minutes and blocked with 10% donkey serum in 0.1% PBS- 0.3% TritonX for 30 minutes at 24°C. The cultured neurons were then stained with ab2723 at 1/1000 in 0.3% TritonX with 0.1x PBS and 10% donkey serum for 4h at 24°C. An Alexa Fluro 568 donkey anti-mouse polyclonal antibody at 1/1000 was used as the secondary antibody. PSD95 (which stains the axons and dendrytic spines) can be observed in red (Alexa fluro 568 secondary). Synaptophysin is observable in green (alexa 488 secondary) and colocalizes with PSD95 in dendritic spines. Nuclei are stained in blue with 1.43µM Hoechst.

This image is courtesy of an Abreview submitted by Ruma Raha-Chowdhury