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).

All lanes : Anti-PML Protein antibody [EPR16792] (ab179466) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : PML knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PML knockout HeLa cell lysate
Lane 3 : 293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PML knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HEK293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 98 kDa
Observed band size: 50-110 kDa why is the actual band size different from the predicted?

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.

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.

All lanes : Anti-PML Protein antibody [EPR16792] (ab179466) at 1/20000 dilution

Lane 1 : 293T (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 4 : A549 (Human lung carcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

All lanes : Anti-PML Protein antibody [EPR16792] (ab179466) at 1/2000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

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 why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

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.