- GTX60929 - GeneTex PTEN antibody

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Supportive validation

Submitted by: GeneTex (provider)
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Experimental details: Western blot analysis of PTEN [pS370] was performed by loading 20 ug of MCF-7 (lane1), HeLa (lane2), NIH/3T3 (lane3), Mouse Brain (lane4), Rat Brain (lane5) and SHSY5Y (lane6) cell lysate using Novex? NuPAGE? 4-12 % Bis-Tris gel, XCell SureLock? Electrophoresis System, Novex? Sharp Pre-Stained Protein Standard, and iBlot? Dry Blotting System. Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. PTEN [pS370] was detected at ~ 58 kDa using PTEN [pS370] Rabbit polyclonal Antibody at 1:1000 dilution in 5 % skim milk at 4¢XC overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:5000 dilution was used and chemiluminescent detection was performed using Novex? ECL Chemiluminescent Substrate Reagent Kit.

Supportive validation

Submitted by: GeneTex (provider)
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Experimental details: Antibody-Peptide Competition and Phosphatase Treatment. Extracts of 3T3-L1 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 3% non-fat dried milk-TBST buffer for one hour at room temperature, and then incubated with the PTEN [pS370] antibody in a 3% non-fat dried milk-TBST buffer for two hours at room temperature, following prior incubation with: the non-phosphopeptide corresponding to the phosphopeptide immunogen (1), a generic phosphoserine-containing peptide (2), the phosphopeptide immunogen (3) or no peptide (4, 5). After washing, the membrane was incubated with goat F(ab"e;)2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignal? method. The data show that only the phosphopeptide corresponding to PTEN [pS370] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.

Supportive validation

Submitted by: GeneTex (provider)
Main image
Experimental details: Immunofluorescence analysis of PTEN [pS370] was done on 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton? X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with PTEN [pS370] Rabbit Polyclonal Antibody at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal? Secondary Antibody, Alexa Fluor? 488 conjugate at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade? Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor? 555 Rhodamine Phalloidin. Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: GeneTex (provider)
Main image
Experimental details: Immunohistochemistry analysis of PTEN showing staining in the cytoplasm and nucleus of paraffin-embedded mouse prostate tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PTEN polyclonal antibody diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4¢XC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: GeneTex (provider)
Main image
Experimental details: Immunohistochemistry analysis of PTEN showing staining in the cytoplasm and nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PTEN polyclonal antibody diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4¢XC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: GeneTex (provider)
Main image
Experimental details: Immunohistochemistry analysis of PTEN showing staining in the cytoplasm and nucleus of paraffin-embedded human prostate carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PTEN polyclonal antibody diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4¢XC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: GeneTex (provider)
Main image
Experimental details: Flow cytometry analysis of PTEN [pS370] was done on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton? X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with PTEN [pS370] Rabbit Polyclonal Antibody (red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor? 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune? Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

GTX60929