重组Anti-PML蛋白抗体[EPR16792]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PML Protein antibody [EPR16792] (AB179466)

ab179466 staining PML in wild-type Hap1 cells (top panel) and PML knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab179466 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PML Protein antibody [EPR16792] (AB179466)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling PML Protein with ab179466 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm and nuclear staining on K562 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows : -
-ve control 1 - ab179466 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PML Protein antibody [EPR16792] (AB179466)

Immunohistochemical analysis of paraffin-embedded Human mammary gland tissue labeling PML Protein with ab179466 at 1/2000 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus staining on epithelial cells of Human mammary gland tissue is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PML Protein antibody [EPR16792] (AB179466)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling PML Protein with ab179466 at 1/2000 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Counter stained with Hematoxylin. The breast cancer cells lost expression.

Reference : Gurrieri C et al. Loss of the Tumor Suppressor PML in Human Cancers of Multiple Histologic Origins. J Natl Cancer Inst 96 : 269 –279 (2004).

Negative control : Used PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-PML Protein antibody [EPR16792] (AB179466)

PML Protein was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell extract with ab179466 at 1/70 diltuion. Western blot was performed from the immunoprecipitate using ab179466 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : K562 whole cell extract. Lane 2 : PBS instead of K562 whole cell extract.

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

ab179466 recognizes 12 isoforms shown as multiple bands ranging from 50 kDa to 110 kDa.

All lanes:

Immunoprecipitation - Anti-PML Protein antibody [EPR16792] (ab179466)

Predicted band size: 98 kDa

Observed band size: 50-110 kDa

false

• WB

Lab

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Lanes 1-3 : Merged signal (red and green). Green - ab179466 observed at 50-110 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab179466 Anti-PML Protein antibody [EPR16792] was shown to specifically react with PML Protein in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261811 (knockout cell lysate ab257081) was used. Wild-type and PML Protein knockout samples were subjected to SDS-PAGE. ab179466 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PML Protein antibody [EPR16792] (ab179466) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PML knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PML (PML Protein) knockout HeLa cell line (<a href='/products/cell-lines/human-pml-pml-protein-knockout-hela-cell-line-ab261811'>ab261811</a>)

Lane 3:

293T cell lysate at 20 µg

Predicted band size: 98 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Lanes 1 - 4 : Merged signal (red and green). Green - ab179466 observed at 50-110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab179466 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in PML knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PML knockout samples were subjected to SDS-PAGE. ab179466 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PML Protein antibody [EPR16792] (ab179466) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PML knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HEK293 whole cell lysate at 20 µg

Predicted band size: 98 kDa

Observed band size: 50-110 kDa

false

• WB

Supplier Data

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab179466 recognizes 12 isoforms shown as multiple bands ranging from 50 kDa to 110 kDa.

All lanes:

Western blot - Anti-PML Protein antibody [EPR16792] (ab179466) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 98 kDa

Observed band size: 50-110 kDa

false

• WB

Supplier Data

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab179466 recognizes 12 isoforms shown as multiple bands ranging from 50 kDa to 110 kDa.

All lanes:

Western blot - Anti-PML Protein antibody [EPR16792] (ab179466) at 1/20000 dilution

Lane 1:

293T (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

A549 (Human lung carcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 98 kDa

Observed band size: 50-110 kDa

false

• WB

Unknown

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Lanes 1-3 : Merged signal (red and green). Green - ab179466 observed at 50-110 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab179466 Anti-PML Protein antibody [EPR16792] was shown to specifically react with PML Protein in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261811 (knockout cell lysate ab257081) was used. Wild-type and PML Protein knockout samples were subjected to SDS-PAGE. ab179466 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PML Protein antibody [EPR16792] (ab179466) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PML knockout HeLa cell lysate at 20 µg

Lane 3:

293T cell lysate at 20 µg

Predicted band size: 98 kDa

Observed band size: 50-110 kDa

false

• WB

Lab

Western blot - Anti-PML Protein antibody [EPR16792] (AB179466)

Lanes 1 - 2 : Merged signal (red and green). Green - ab179466 observed at 110 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab179466 was shown to react with PML in A549 wild-type cells in western blot with loss of signal observed in PML knockout cell line ab266980 (PML knockout cell lysate ab257082). Wild-type and PML knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk before incubation with ab179466 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 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-PML Protein antibody [EPR16792] (ab179466) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

PML knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human PML (PML Protein) knockout A549 cell line (<a href='/products/cell-lines/human-pml-pml-protein-knockout-a549-cell-line-ab266980'>ab266980</a>)

Secondary

All lanes:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (<a href='/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='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 98 kDa

Observed band size: 110 kDa

false