Anti-P2X4 antibody [EPR29153-16]

• ICC

Supplier Data

Immunocytochemistry - Anti-P2X4 antibody [EPR29153-16] (AB324165)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling P2X4 with ab324165 at 1/50 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 T-47D and no staining in HeLa cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : HeLa.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-P2X4 antibody [EPR29153-16] (AB324165)

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

This antibody does not cross-react with human P2RX3 or P2RX5.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-P2X4 antibody [EPR29153-16] (ab324165) at 1/1000 dilution

Lane 1:

P2RX4 knockout HEK-293 transfected with a human P2RX4 expression vector containing a His-tag

Lane 2:

P2RX4 knockout HEK-293 transfected with a human P2RX3 expression vector containing a His-tag

Lane 3:

P2RX4 knockout HEK-293 transfected with a human P2RX5 expression vector containing a His-tag

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

Observed band size: 55 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-P2X4 antibody [EPR29153-16] (AB324165)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-P2X4 antibody [EPR29153-16] (ab324165) at 1/1000 dilution

Lane 1:

Mouse dorsal galion tissue lysate at 20 µg

Lane 2:

Rat dorsal galion tissue lysate at 20 µg

Lane 3:

Mouse bladder tissue lysate at 20 µg

Lane 4:

Rat bladder tissue lysate at 20 µg

Lane 5:

Human bladder tissue 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: 55 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-P2X4 antibody [EPR29153-16] (AB324165)

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

Performed under reducing conditions.

In Western blot, ab324165 was shown to bind specifically to P2X4. Target of interest was observed at 55 kDa in HEK-293T cell lysates (lane 1) with no signal observed at this size in P2X4 knockout cell line (lane 2) (lane 2, knockout cell line ab266170 / knockout cell lysate ab258089).

Low expression : HeLa, F9

Lane1 and 2 were developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane1-2 : 59 seconds, lane3-4 : 15 seconds, lane5-6 : 180 seconds

All lanes:

Western blot - Anti-P2X4 antibody [EPR29153-16] (ab324165) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

P2RX4 knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

N9 (mouse microglia cell) whole cell lysate at 20 µg

Lane 6:

F9 (mouse embryonal carcinoma epithelial 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 1/100000 dilution

Observed band size: 55 kDa,36 kDa

true

• WB

Supplier Data

Western blot - Anti-P2X4 antibody [EPR29153-16] (AB324165)

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

Low expression : brain(PMID : 12845522)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-P2X4 antibody [EPR29153-16] (ab324165) at 1/1000 dilution

Lane 1:

Mouse pancreas tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat pancreas tissue lysate at 20 µg

Lane 4:

Rat colon tissue lysate at 20 µg

Lane 5:

Rat brain tissue 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

Observed band size: 55 kDa,36 kDa

false

Exposure time: 59s