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Western blot
ImmunoprecipitationAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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- Product number
- PA1-16526 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- PHD3 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Suggested positive control: antigen standard for EGLN3 (transient overexpression lysate).
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 1 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 detection of Human and Rat PHD3. Samples: (A) Lysate from cells that were transiently transfected with PHD1, PHD2, PHD3 or PHD4 constructs or in vitro translated PHD1, PHD2, PHD3, PHD4. (B) Lysate from rat cells before or 9 hours after inducing hypoxia. Antibody: Affinity purified rabbit anti-PHD3.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-PHD3 Polyclonal Antibody (Product # PA1-16526) and a 27 kDa band corresponding to PHD3 was observed across cell lines tested except U-937 and BeWo which are reported to be negative. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), MCF-7 (Lane 2), U-937 (Lane 3) and BeWo (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (1:2000 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 PHD3 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 PHD3 Rabbit Polyclonal Antibody (Product # PA1-16526) at 1:250 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 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 Cytoplasmic 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
- Immunohistochemical analysis of PHD3 in formalin fixed tissue section of human renal cell carcinoma (clear cell type). Samples were incubated in PHD3 polyclonal antibody (Product # PA1-16526) followed by HRP-DAB detection and hematoxylin counterstaining. The antibody generated a strong nuclear staining of PHD3 primarily in the cancer cells while the stromal cells were largely negative for this protein.
