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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
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- Product number
- PA5-28859 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- EphB4 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: 293T, A431, Jurkat, Raji.
- Concentration
- 0.63 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using EphB4 Polyclonal Antibody (Product # PA5-28859). Sample (30 µg of whole cell lysate). Lane A: A431 . 7.5% SDS PAGE. Eph receptor B4 antibody . EphB4 Polyclonal Antibody (Product # PA5-28859) diluted at 1:1,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-EphB4 Polyclonal Antibody (Product # PA5-28859) and a 108 kDa band corresponding to EPHB4 was observed cross cell lines tested except A549 and U-87 MG which are reported to be negative. Whole cell extracts (30 µg lysate) of NIH:OVCAR3 (Lane 1), Caco-2 (Lane 2), HT-29 (Lane 3), A549 (Lane 4) and U-87 MG (Lane 5) 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 SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of EphB4 was performed using 70% confluent log phase HT-29 and A549 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 EphB4 Rabbit Polyclonal Antibody (Product # PA5-28859) at 5 µg/mL 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 (Product # A27034) 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 A549 cells having no expression of EphB4. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
