All lanes : Anti-PTEN antibody [EPR22636-122] (ab267787) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 5 : Mouse kidney tissue lysate
Lane 6 : Mouse spleen tissue lysate
Lane 7 : Rat lung tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 47 kDa
Observed band size: 54 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times:

Lanes 1-6: 3 seconds;
Lane 7: 7 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID:10514407).

All lanes : Anti-PTEN antibody [EPR22636-122] (ab267787) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PTEN knockout HAP1 whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : MDA-MB-468 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 47 kDa
Observed band size: 54 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times:

Lanes 1-4: 3 seconds;
Lane 5: 114 seconds.

ab267787 was shown to specifically react with PTEN in wild-type HAP1 cells as signal was lost in PTEN knockout cells. Wild-type and PTEN knockout samples were subjected to SDS-PAGE. Ab267787 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/20,000 dilution for 1 hour at room temperature before imaging.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:10514407).

Negative control: MDA-MB-468 (PMID:21358673,15674339).

 

 

PTEN was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab267787 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267787 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab267787 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267787 in HeLa whole cell lysate

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

Exposure time: 15 seconds

 

PTEN was immunoprecipitated from 0.35 mg MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate with ab267787 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267787 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab267787 IP in MCF7 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267787 in MCF7 whole cell lysate

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

Exposure time: 15 seconds

 

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling PTEN with ab267787 at 1/2000 dilution (2.18 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat pancreas (PMID:11021813). The section was incubated with ab267787 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling PTEN with ab267787 at 1/2000 dilution (2.18 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse pancreas (PMID:11021813). The section was incubated with ab267787 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling PTEN with ab267787 at 1/2000 dilution (2.18 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human pancreas (PMID:11021813). The section was incubated with ab267787 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling PTEN with ab267787 at 1/2000 dilution (2.18 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stroma of human ovarian cancer (PMID:25608477). The section was incubated with ab267787 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human endometrial cancer tissue labeling PTEN with ab267787 at 1/2000 dilution (2.18 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stroma of human endometrial cancer (PMID:2230170). The section was incubated with ab267787 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling PTEN with ab267787 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling PTEN with ab267787 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.