All lanes : Anti-PSD95 antibody [EPR23124-118] (ab238135) at 1/2000 dilution

Lane 1 : Human cerebellum lysate
Lane 2 : Human brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 80 kDa
Observed band size: 100,75 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 20118925)

Exposure time: 48 seconds

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat retina tissue labeling VSIG4 with ab238135 at 1/100 dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Rat retina tissue labeling PSD95 with ab238135 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on outer plexiform layer of rat retina is observed. The section was incubated with ab238135 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins.

PSD95 was immunoprecipitated from 0.35 mg Mouse brain lysate with ab238135 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab238135 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain lysate 10µg
Lane 2: ab238135 IP in Mouse brain lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab238135 in Mouse brain lysate

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

Exposure time: 3 seconds.

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Mouse retina tissue labeling VSIG4 with ab238135 at 1/100 (5.76 µg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution.

All lanes : Anti-PSD95 antibody [EPR23124-118] (ab238135) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse cerebellum lysate
Lane 3 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 80 kDa
Observed band size: 100,75 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 20118925).

Exposure time: 15 seconds

Immunohistochemical analysis of paraffin-embedded Mouse cerebellum tissue labeling PSD95 with ab238135 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on basket cells of mouse cerebellum is observed. The section was incubated with ab238135 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins.