Anti-CD2AP antibody [EPR30265-529]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEL (human erythroleukemia erythroblast) and U-118 MG (human brain glioblastoma cell) cells labelling CD2AP with ab325800 at 1/50 (10.2 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in HEL cell line and no staining in U-118 MG cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : U-118 MG.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325800 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CD2AP with ab325800 at 1/500 (1.02 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling CD2AP with ab325800 at 1/500 (1.02 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human kidney (PMID : 15149332).

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human kindey tissue staining CD2AP with ab325800 at a 1 : 500 (1.02 µg/ml) dilution, ab273021 anti-V2R used at 1 : 100 (6.32 µg/ml) dilution and ab259976 anti-Synaptopodin used at a 1 : 500 (0.934 µg/ml) dilution.

Panel A : anti-CD2AP (green; Opal™520), anti-V2R (magenta; Opal™570), anti-Synaptopodin (yellow; Opal™690) on human kidney.
Panel B : anti-CD2AP showed positive staining in human kidney.
Panel C : anti-V2R staining connecting ducts in human kidney.
Panel D : anti-Synaptopodin staining glomeruli in human kidney.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325800, ab273021 and ab259976 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded
(A) K-562 (human chronic myelogenous leukemia lymphoblast) cell pellet
(B) HEL (human erythroleukemia erythroblast) cell pellet
(C) RD (human muscle multinucleated spindle-shaped cell) cell pellet
labeling CD2AP with ab325800 at 1/4000 (0.128 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining (A) K-562 (human chronic myelogenous leukemia lymphoblast) cell pellet and (B) HEL (human erythroleukemia erythroblast) cell pellet, no staining on (C) RD (human muscle multinucleated spindle-shaped cell) cell pellet.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling CD2AP with ab325800 at 1/500 (1.02 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human gastric adenocarcinoma.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling CD2AP with ab325800 at 1/500 (1.02 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : Weak staining on endothelial cells of human cerebrum (PMID : 34514662).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-CD2AP antibody [EPR30265-529] (AB325800)

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

Low expression : RD.

The identity of the bands lower than 60 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 10 seconds; Lanes 3-4 : 59 seconds.

All lanes:

Western blot - Anti-CD2AP antibody [EPR30265-529] (ab325800) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HEK-293 transfected with siRNA specifically targeting CD2AP whole cell lysate at 20 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

RD (human muscle multinucleated spindle-shaped cell) whole cell 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 1/20000 dilution

Observed band size: 80 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-CD2AP antibody [EPR30265-529] (AB325800)

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

Low expression : U-118 MG.

The identity of the bands lower than 60 kDa are unknown.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD2AP antibody [EPR30265-529] (ab325800) at 1/1000 dilution

Lane 1:

HEL (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 2:

U-118 MG (human brain glioblastoma cell) whole cell 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 1/20000 dilution

Observed band size: 80 kDa,36 kDa

false

Exposure time: 8s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD2AP antibody [EPR30265-529] (AB325800)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a CD2AP expression vector containing a his tag and (B) HEK-293T cells transfected with empty vector containing a myc-His-tag tissue labeling CD2AP with ab325800 at 1/15000 (0.034 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining (A) HEK-293T transfected with a CD2AP expression vector containing a his tag, no staining on (B) HEK-293T cells transfected with empty vector containing a myc-His-tag.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins