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

Lanes 1-6 : Anti-PSD95 antibody [6G6-1C9] (ab2723) at 1/1200 dilution
Lanes 7-12 : Anti-PSD95 antibody [6G6-1C9] (ab2723) at 1/1200 dilution (18 hours at 4°C)

Lanes 1-6 : Mouse Hippocampus with Li-Cor Block Buffer, 1 hour at 21°C
Lanes 7-12 : Mouse Liver with Li-Cor Block Buffer, 1 hour at 21°C

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Li-Cor IRDye® Donkey anti-mouse 680LT at 1/18000 dilution

Performed under reducing conditions.

Predicted band size: 80 kDa
Additional bands at: 95 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 5 minutes

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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.

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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
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (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

ab2723 staining PSD95 in Zebrafish retina (outer plexiform layer) tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 5% BSA for 1 hour at 23°C. Sodium citrate antigen retrieval was used. Samples were incubated with primary antibody (1/100) for 16 hours at 4°C. An Alexa Fluor^® 647-conjugated goat anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

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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.

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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

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IHC-FoFr staining of PSD95 on rat hippocampus section using ab2723 (1:3000). The sections used came from animals perfused fixed with Paraformaldehyde 4% with 15% of a solution of saturated picric acid, in phosphate buffer 0.1M. Following postfixation in the same fixative overnight, the brains were cryoprotected in sucrose 30% overnight. Brains were then cut using a cryostat and the immunostainings were performed using the ‘free floating’ technique.

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