Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker

9 product images

• 

  Western blot - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  Lanes 1 - 4: Merged signal (red and green). Green - ab238135 observed at 75 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
  

  ab238135 was shown to react with PSD95 in wild-type SH-SY5Y cells in Western blot with loss of signal observed in DLG4 knockout cell line Human PSD95 knockout SH-SY5Y cell line ab280043 (DLG4 knockout cell lysate Human PSD95 knockout SH-SY5Y cell lysate ab280102). Wild-type SH-SY5Y and DLG4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab238135 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

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

  Lane 1: Wild-type SH-SY5Y cell lysate at 20 µg

  Lane 2: DLG4 knockout SH-SY5Y cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: HL-60 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 80 kDa

  Observed band size: 75 kDa

• 

  Western blot - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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

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

  Lane 1: Human cerebellum lysate at 20 µg

  Lane 2: Human brain lysate at 20 µg

  Secondary

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

  Predicted band size: 80 kDa

  Observed band size: 100 kDa, 75 kDa

• 

  Immunoprecipitation - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab238135 in Mouse brain lysate

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

  Exposure time: 3 seconds.

  All lanes: Immunoprecipitation - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  Predicted band size: 47 kDa, 80 kDa

• 

  Immunohistochemistry (Frozen sections) - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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 (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 (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.

• 

  Immunohistochemistry (Frozen sections) - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution.

• 

  Western blot - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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

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

  Lane 1: Mouse brain lysate at 20 µg

  Lane 2: Mouse cerebellum lysate at 20 µg

  Lane 3: Rat brain lysate at 20 µg

  Secondary

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

  Predicted band size: 80 kDa

  Observed band size: 100 kDa, 75 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  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 (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 (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.

• 

  Western blot - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (ab238135)

  Western blot: Anti-DLG4 antibody [EPR23124-118] (ab238135) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab238135 was shown to bind specifically to DLG4. A band was observed at 80 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in DLG4 knockout cell line. To generate this image, wild-type and DLG4 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

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

  Lane 1: Wild-type U-87 MG cell lysate at 20 µg

  Lane 2: DLG4 knockout U-87 MG cell lysate at 20 µg

  Lane 3: Wild-type SH-SY5Y cell lysate at 20 µg

  Lane 4: DLG4 knockout SH-SY5Y Human PSD95 knockout SH-SY5Y cell line ab280043 cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.