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
- 711337 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-AS160 (Ser588) Recombinant Polyclonal Antibody (5HCLC)
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Bovine, Mouse
- Antibody clone number
- 5HCLC
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Insulin (30 nmol/L for 15 mins) (Lane 2) and HeLa treated with IGF1 (100 ng/mL for 15 mins). The blots were probed with Anti-AS160 [pS588] Recombinant Rabbit Polyclonal Antibody (Product # 711337, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 160 kDa band corresponding to AS160 [pS588] was observed across the cell lines tested. 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 by wet transfer method. 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 HT-29 cells were fixed and permeabilized for detection of endogenous AS160[pS588] using Anti-AS160[pS588] Recombinant Rabbit Polyclonal Antibody (Product # 711337, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of AS160[Ps588] (green) in the cytoplasm can be clearly observed in cells treated with IGF (100 ng/mL, 15 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the AS160[Ps588] phospho peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.