Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Chromatin Immunoprecipitation [2]
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Validation data
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- Product number
- PA5-41514 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CTCF Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Peptide sequence: MEGDAVEAIV EESETFIKGK ERKTYQRRRE GGQEEDACHL PQNQTDGGEV Sequence homology: Cow: 100%; Dog: 100%; Guinea Pig: 100%; Horse: 100%; Human: 100%; Mouse: 100%; Rabbit: 100%; Rat: 100%
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 0.5 mg/mL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of transfected 293T cells using an anti-CTCF polyclonal antibody (Product # PA5-41514).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of CTCF was achieved by transfecting MCF7 with CTCF specific siRNAs (Silencer® select Product # s20966, s20967). Western blot analysis (Fig. a) was performed using nuclear enriched extracts from the CTCF knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CTCF Polyclonal Antibody (Product # PA5-41514, 0.5 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 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 CTCF.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-CTCF Polyclonal Antibody (Product # PA5-41514) and a 110 kDa band corresponding to CTCF was observed across cell lines tested. Nuclear enriched extracts (30 µg lysate) of MCF7 (Lane 1), HeLa (Lane 2), Hep G2 (Lane 3), LNCaP (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580). Additional uncharacterized band was observed at ~60 kDa.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of CTCF was performed using 70% confluent log phase MCF7 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 1 hour at room temperature. The cells were labeled with CTCF Polyclonal Antibody (Product # PA5-41514) 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 nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- ChIP assay analysis of HCT116 cells using an anti-CTCF polyclonal antibody (Product # PA5-41514).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Chromatin Immunoprecipitation (ChIP) of CTCF protein was performed using CTCF Polyclonal Antibody (Product # PA5-41514, 5 µg) on sheared chromatin from MCF7 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to H19-ICR, CRY1, PFKFB3, GLI2 (5'), AXL (3') (Active), EXON4/5 OF H19 and SAT2 satellite repeats (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.