Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [2]
- Immunohistochemistry [4]
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- Product number
- MA5-25368 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Gephyrin Monoclonal Antibody (OTI3B6)
- Antibody type
- Monoclonal
- Antigen
- Recombinant full-length protein
- Reactivity
- Human, Mouse, Rat
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- OTI3B6
- Vial size
- 100 µL
- Concentration
- 1 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 GPHN in HepG2, HeLa, HT29, A549, COS7, Jurkat, MDCK, PC12, MCF7 cells using 35 µg per lane. Samples were probed with GPHN (Product # MA5-25368) monoclonal antibody.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of GPHN was achieved by transfecting PC-3 with GPHN specific siRNAs (Silencer® select Product # s20000, s20001). Western blot analysis (Fig. a) was performed using whole cell extracts from GPHN knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Gephyrin Monoclonal Antibody (OTI3B6) (Product # MA5-25368, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 GPHN..
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-Gephyrin Monoclonal Antibody (OTI3B6) (Product # MA5-25368) and 93 kDa, 155 kDa and 250 kDa bands corresponding to GPHN was observed across all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of IMR32 (Lane 1), Neuro-2a (Lane 2), PC-3 (Lane 3), PC-12 (Lane 6) and tissue extracts of Rat Brain (Lane 4) and Mouse Brain (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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). The 155 kDa and 250 kDa bands observed in PC-12 are reported to be SUMOylated form of GPHN. [doi: 10.1038/ncomms13365 (2016)] and the 25 kDa band in Mouse Brain is the circulating IgGs that are commonly detected in tissues..
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of GPHN in COS7 cells. Cells were transfected with a plasmid overexpressing GPHN and probed with a GPHN monoclonal antibody (Product # MA5-25368).
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescence analysis of Gephyrin (GPHN) was performed using PC-3 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 Gephyrin Monoclonal Antibody (OTI3B6) (Product # MA5-25368) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723, 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 punctate nuclear staining of GPHN. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human ovary tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a GPHN monoclonal antibody (Product # MA5-25368).
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunohistochemistry was performed on paraffin-embedded human prostate tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a GPHN monoclonal antibody (Product # MA5-25368).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry was performed on paraffin-embedded human liver tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a GPHN monoclonal antibody (Product # MA5-25368).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human colon tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a GPHN monoclonal antibody (Product # MA5-25368).