Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [2]
- Immunocytochemistry [1]
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Validation data
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- Product number
- PA5-32374 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Anti-Cyclin E Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with porcine based on sequence homology. 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 10 min at room temperature. A suggested positive control is breast carcinoma or placenta tissue.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 500 µL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of Cyclin E was achieved by transfecting A549 with Cyclin E specific siRNAs (Silencer® select Product # S2525, S2526). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Cyclin E knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cyclin E Polyclonal Antibody (Product # PA5-32374, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 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 Cyclin E.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-Cyclin E Polyclonal Antibody(Product # PA5-32374) and a 48kDa band corresponding to Cyclin E was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), K-562 (Lane 3) and Hep G2 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 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).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Cyclin E was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Cyclin E Polyclonal Antibody (Product # PA5-32374) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.