Anti-p15 INK4b antibody [EPR29010-111]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-p15 INK4b antibody [EPR29010-111] (AB323851)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell) cells labelling p15 INK4b with ab323851 at 1/100 (5.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in MCF7 cells (shown in green) treated with TGFβ (10 ng/mL) for 18 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). 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/ab195889 200 2.5ug/ml 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/ab150081 1000 2ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-p15 INK4b antibody [EPR29010-111] (AB323851)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 12202478).

The expression of p15 INK4b is upregulated in response to TGF beta treatment (PMID : 12202478).

The identity of the bands higher than 15 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) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-p15 INK4b antibody [EPR29010-111] (ab323851) at 1/1000 dilution

Lane 1:

Untreated MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 treated with 10ng/ml TGF beta 1 for 18 hours 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: 14-15 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-p15 INK4b antibody [EPR29010-111] (AB323851)

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

Negative control : THP-1

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 12202478).

The identity of the bands higher than 15 kDa in lane 1 are unknown.

To minimize protein degradation, cells in lanes 1-2 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) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-p15 INK4b antibody [EPR29010-111] (ab323851) at 1/1000 dilution

Lane 1:

HaCaT (human skin keratinocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

Calu-1 (human lung epidermoid 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 1/20000 dilution

Observed band size: 14-15 kDa,36 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-p15 INK4b antibody [EPR29010-111] (AB323851)

Dot blot analysis of p15 INK4b using ab323851 at 1 : 1000 (0.508 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : Human p15 INK4b peptide
Lane2 : Human CDKN2A/p16INK4a peptide

Exposure time : 180 seconds.

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

This antibody does not cross-react with human CDKN2A/p16INK4a.

All lanes:

Dot Blot - Anti-p15 INK4b antibody [EPR29010-111] (ab323851) at 1/1000 dilution

Lane 1:

Human p15 INK4

Lane 2:

Human CDKN2A/p16INK4a peptide

Secondary

All lanes:

Dot 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

Exposure time: 180s