- 44-640G - Invitrogen Antibodies TP53 antibody

Huntington ND, Xu Y, Nutt SL, Tarlinton DM
The Journal of experimental medicine 2005 May 2;201(9):1421-33

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition. HEK 293 extracts were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either untreated (1-4) or treated with 4000 units/mL lambda phosphatase at 37oC for 1 hour (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, and then incubated with the p53 (pS392) antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphoserine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to p53 (pS392) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane 1), A549 (Lane 2), A549 treated overnight with 250 mM of Doxorubicin (lane 3), HEK-293 (lane 4) and HEK-293 treated for 6 hr with 25 uM of Etoposide (lane 5). The blots were probed with Anti-Phospho-p53 (Ser392) Rabbit Polyclonal Antibody (Product # 44-640G, 1:250 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 53 kDa band corresponding to p53 (pS392) was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane 1), A549 (Lane 2), A549 treated overnight with 250 mM of Doxorubicin (lane 3), HEK-293 (lane 4) and HEK-293 treated for 6 hr with 25 uM of Etoposide (lane 5). The blots were probed with Anti-Phospho-p53 (Ser392) Rabbit Polyclonal Antibody (Product # 44-640G, 1:250 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 53 kDa band corresponding to p53 (pS392) was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of P53 (PS392) showing staining in the nucleus and weakly in the cytoplasm of paraffin-embedded human pancreatic 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 P53 (PS392) Rabbit Polyclonal Antibody (Product # 44-640G) diluted in 3% BSA-PBS at a dilution of 1:20 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 p53 [pSer392] was done on A-431 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 p53 [pSer392] Rabbit Polyclonal Antibody (44640G, 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 (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.

44-640G