Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
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Validation data
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- Product number
- 711152 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-WEE1 (Ser642) Recombinant Polyclonal Antibody (11HCLC)
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 11HCLC
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Nuclear enriched extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with EGF (100nM for 10min) (Lane 2), HEK-293 (Lane 3) and HEK-293 treated with EGF (100nM for 10min) (Lane 4). The blots were probed with Anti-Wee1 (pS642) Recombinant Rabbit Polyclonal Antibody (Product # 711152, 1-2 µg/mL) 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 95 kDa band corresponding to Wee1 (pS642) was observed. To confirm the specificity of Anti-Wee1 (pS642) Recombinant Rabbit Polyclonal Antibody, competition was performed with the posphopeptide (10 µg/mL) as shown in the corresponding blot on the right. The phosphopeptide competes with the antibody and prevents it from binding to the target protein. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), 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® 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
- For immunofluorescence analysis, A-431 cells were fixed and permeabilized for detection of endogenous Wee1 (pS642) using Wee1 (pS642) Recombinant Rabbit Polyclonal Antibody (Product # 711152, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Wee1 (pS642) protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Wee1 (pS642). Panel e) shows loss of signal by competition with the Wee1 (pS642) phosphopeptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phospho peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.