Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

This data was developed using ab238135, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling PSD95 with ab238135 at 1ug/ml followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining in human Alzheimer's brain.

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 LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

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. 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab238135).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

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. 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab238135).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat retina tissue labeling VSIG4 with ab238135 at 1/100 (5.76 μg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/000 (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab238135).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab238135).

• IP

Unknown

Immunoprecipitation - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab238135).

All lanes:

Immunoprecipitation - Anti-PSD95 antibody [EPR23124-118] - Synaptic Marker (<a href='/en-us/products/primary-antibodies/psd95-antibody-epr23124-118-synaptic-marker-ab238135'>ab238135</a>)

Predicted band size: 47 kDa,80 kDa

false

• WB

Lab

Western blot - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

This data was developed using ab238135, the same antibody clone in a different buffer formulation.

Western blot : Anti-DLG4 antibody [EPR23124-118] (ab238135) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (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 (<a href='/en-us/products/primary-antibodies/psd95-antibody-epr23124-118-synaptic-marker-ab238135'>ab238135</a>) at 1/2000 dilution

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

Western blot - Human DLG4 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-dlg4-knockout-u-87-mg-cell-line-ab306652'>ab306652</a>)

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 <a href='/en-us/products/cell-lines/human-psd95-knockout-sh-sy5y-cell-line-ab280043'>ab280043</a> 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

false

• WB

Unknown

Western blot - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (ab269863) 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) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 80 kDa

Observed band size: 100 kDa,75 kDa

false

• WB

Lab

Western blot - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

This data was developed using the same antibody clone in a different buffer formulation (ab238135).

Lanes 1 - 4 : Merged signal (red and green). Green - ab238135 observed at 75 kDa. Red - 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 ab280043 (DLG4 knockout 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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 (<a href='/en-us/products/primary-antibodies/psd95-antibody-epr23124-118-synaptic-marker-ab238135'>ab238135</a>) 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 2:

Western blot - Human PSD95 knockout SH-SY5Y cell line (<a href='/en-us/products/cell-lines/human-psd95-knockout-sh-sy5y-cell-line-ab280043'>ab280043</a>)

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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 80 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-PSD95 antibody [EPR23124-118] - BSA and Azide free (AB269863)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false