Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
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Validation data
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- Product number
- PA5-32577 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Anti-S6 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 30 min at room temperature. A suggested positive control is breast carcinoma.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 500 µL
- 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
- Knockdown of S6 was achieved by transfecting HeLa with S6 specific siRNAs (Silencer® select Product # S12270, S12272). Western blot analysis (Fig. a) was performed using whole cell extracts from the S6 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with S6 Polyclonal Antibody (Product # PA5-32577, 1:25 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:2000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to S6.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-S6 Polyclonal Antibody(Product # PA5-32577) and a 31kDa band corresponding to S6 was observed across all cell lines tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), HeLa (Lane 2), Hep G2 (Lane 3), Jurkat (Lane 4), NTERA2 cl.D1 (Lane 5), U-87 MG (Lane 6) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:25 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of NIH3T3 Cells using anti-S6 Ribosomal Polyclonal Antibody (Product # PA5-32577). The recommened dilution for this antibody in western blot applications is 1:25.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of S6 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with S6 Polyclonal Antibody (Product # PA5-32577) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytosolic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.