Total proteins were lysed by RIPA buffer (added with protease inhibitors) on ice and then were centrifuged at 12,000 × g at 4°C for 5 min. A mixture of equal protein and sample buffer was subjected to 15% SDS-PAGE. The separated protein was then transferred onto a PVDF membrane (Millipore Corporation, Billerica, MA, USA). The PVDF membrane was blocked in blocking buffer (containing TBST and 5% fat-free milk) for 1 h at room temperature. Primary antibody (rabbit anti-PTEN antibody, 1:1,000) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH acted as the internal reference. PVDF membrane was incubated in the diluted primary antibodies at 4°C overnight. The PVDF membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:500; Abcam, Cambridge, MA, USA) at 4°C overnight. Immunoblots were visualized by ECL kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scanned by ImageJ software. The band densities of each sample were normalized to the GAPDH band.
Rabbit anti pten antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit anti-PTEN antibody is a primary antibody that specifically detects the PTEN protein. PTEN is a tumor suppressor protein that plays a role in the regulation of cell growth and division. The antibody can be used to study the expression and localization of PTEN in various cellular and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Ecl kit, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Abcam (1 mentions)
Protein a sepharose beads, by Merck Group (1 mentions)
Malachite green assay kit, by Echelon Biosciences (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p53 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti mdm2 antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
5 protocols using rabbit anti pten antibody
1
PTEN Protein Expression Analysis
2
PTEN Phosphatase Activity Assay
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MM and ULM cells were cultured in 100-mm dishes in complete DMEM-F12 1:1 medium until 80 to 90% confluence. Cells were lysed in ice-cold 25 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 5% glycerol lysis buffer. PTEN (500 μg) was immunoprecipitated from whole-cell lysates with 8 μl of rabbit anti-PTEN antibody (Cell Signaling Technologies) under agitation at 4°C overnight. The antigen-antibody complex was recovered with 30 μl of protein A Sepharose beads (Sigma-Aldrich) after 3 hours with agitation at 4°C, and then the beads were washed twice in lysis buffer, washed once in PTEN reaction buffer [25 mM tris-HCl (pH 7.4), 140 mM NaCl, 2.7 mM KCl, and 10 mM DTT], and resuspended in 80 μl of PTEN reaction buffer. PTEN phosphatase activity was measured using the Malachite Green Assay Kit (Echelon Biosciences) and PIP3 as substrate according to the manufacturer’s instructions.
3
Immunofluorescent Localization of PTEN
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After 7 or 14 days in vitro, the medium was removed from the plate, and the coverslips were washed twice with cold PBS. The cells were fixed in 4% formaldehyde for 15 min at 4 °C and incubated in blocking solution (5% donkey serum; 0.01% Triton X-100 in PBS) for 1 h at 25 °C prior to overnight incubation with rabbit anti-PTEN antibody (1:200, Cell Signaling Technology, #138G6) at 4 °C. The cells were washed with PBS three times for 10 min each time and then incubated with anti-rabbit Alexa Fluor 488 (1:1000, Life Technology, #A21206) for 2 h at 25 °C. DAPI (1:25,000) was used to stain nuclei. Images were captured under a fluorescence microscope (Nikon 80i, Imaging Software–NIS Element V4.6) using a 20× objective and analyzed with ImageJ (NIH) software.
4
PTEN Immunofluorescence Staining
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24 h after transfection, cells were fixed using 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% triton x-100 in PBS, blocked with 10% BSA, and incubated overnight with rabbit anti-PTEN antibody (138G6, Cell Signaling Technology). Coverslips were then incubated with mouse anti-rabbit Alexa Fluor 568-conjugated antibody (Invitrogen), followed by DAPI, and mounted using ProLong Gold antifade mountant (Thermo Fisher Scientific).
5
Protein Expression Analysis via Western Blot
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We used RIPA lysis buffer (CW Biotech, Beijing, China) to lyse the cells to extract total protein and measure protein concentration by BCA protein assay kit (Thermo Fisher Scienti c, Waltham, MA, USA). To separate protein samples in equal amounts, 10% SDS-PAGE must be used. Next, we transferred and blocked the complex onto a nitrocellulose membrane (General Electric Co, USA). After blocked with 5% skim milk, the blots were probed with primary antibodies. Following washed with PBS, the horseradish peroxidase-conjugated Goat Anti-Rabbit IgG secondary antibodies (ab205718, 1 2, 000) were added.
Next, we incubated the membrane for 1 hour at room temperature. The primary antibodies used in this study including Rabbit Anti-DMTF1 antibody (ab246945, 1:500), Rabbit Anti-PTEN antibody (ab32199, 1:500) , Rabbit Anti-p53 antibody (ab32389, 1:100), Rabbit Anti-MDM2 antibody (ab38618, 1:400) and Rabbit Anti-ARF antibody (ab77581, 1:300) (Cell Signaling Technology, Inc. Danvers, MD, USA). We then uniformly added the chemiluminescence reagent to the lm and developed the image with a developer. We used Tubulin as an endogenous control to normalize protein expression. The fold change versus control group was the demonstration of target proteins' relative expression levels.
Next, we incubated the membrane for 1 hour at room temperature. The primary antibodies used in this study including Rabbit Anti-DMTF1 antibody (ab246945, 1:500), Rabbit Anti-PTEN antibody (ab32199, 1:500) , Rabbit Anti-p53 antibody (ab32389, 1:100), Rabbit Anti-MDM2 antibody (ab38618, 1:400) and Rabbit Anti-ARF antibody (ab77581, 1:300) (Cell Signaling Technology, Inc. Danvers, MD, USA). We then uniformly added the chemiluminescence reagent to the lm and developed the image with a developer. We used Tubulin as an endogenous control to normalize protein expression. The fold change versus control group was the demonstration of target proteins' relative expression levels.
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