Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunohistochemistry [3]
- Flow cytometry [1]
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Validation data
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- Product number
- 44-1062G - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-PTEN (Thr383) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blotting Experiments. Lysates prepared from 3T3-L1 cells (Figure 1, Lanes 1-4), or HEK293 transiently transfected with wild-type PTEN (Figure 2, Lane 1), PTEN T382A (Figure 2, Lane 2), PTEN T383A (Figure 2, Lane 3), or PTEN S385A mutant (Figure 2, Lane 4), were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with PTEN (pT383) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (Figure 1, Lane 1), the non-phosphopeptide corresponding to the immunogen (Figure 1, Lane 2), a generic phosphothreonine-containing peptide (Figure 1, Lane 3), the phosphopeptide immunogen (Figure 1, Lane 4). After washing, membranes were incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method.The data show that only the peptide corresponding to PTEN (pT383) completely blocks the antibody signal, and that the signal is lost only in the T383A mutant (provided by Dr. Alan Hall), verifying that the antibody is site phospho-specific.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of Phospho-PTEN (pThr383) showing staining in the cytoplasm and nucleus of paraffin-embedded human brain 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 Anti- Phospho-PTEN (pThr383) Polyclonal Antibody (Product # 44-1062G) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of Phospho-PTEN (pThr383) 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 Anti- Phospho-PTEN (pThr383) Polyclonal Antibody (Product # 44-1062G) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of Phospho-PTEN (pThr383) showing staining in the cytoplasm and nucleus of paraffin-embedded mouse brain 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 Anti- Phospho-PTEN (pThr383) Polyclonal Antibody (Product # 44-1062G) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C 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
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of Phospho-PTEN [pThr383] 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 Phospho-PTEN [pThr383] Rabbit Polyclonal Antibody (441062G, red histogram) or with rabbit isotype control (yellow 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 (A11008) 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.