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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
- 703746 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- USP14 Recombinant Rabbit Monoclonal Antibody (23H2L11)
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- This antibody is predicted to react with Rat, Bovine.
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 23H2L11
- Vial size
- 100 µg
- Concentration
- 0.5 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
- Knockdown of USP14 was achieved by transfecting LNCaP cells with USP14 specific validated siRNA (Silencer® select Product # s17358). Western blot analysis (Fig a) was performed using membrane enriched extracts from USP14 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-USP14 Recombinant Rabbit Monoclonal Antibody (Product # 703746) at a 1:1000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a 1:5000 dilution. Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that the antibody is specific to USP14.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-USP14 Recombinant Rabbit Monoclonal Antibody (Product # 703746) and a 56 kDa band corresponding to USP14 was observed across the cell lines tested. Membrane enriched extracts (30 µg lysate) of LNCaP (Lane 1), DU145 (Lane 2), PC-3 (Lane 3), MCF7 (Lane 4), MDA-MB-231 (Lane 5), T-47D (Lane 6), A549 (Lane 7), Hep G2 (Lane 8) and Neuro-2a (Lane 9) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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 Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a 1:5000 dilution. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunocytochemistry analysis, MCF-7 cells were fixed and permeabilized using ice cold 100% Acetone for detection of endogenous USP14 using Anti-USP14 Recombinant Rabbit Monoclonal Antibody (Product # 703746) at a 1:100 dilution and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a 1:2000 dilution. Panel a) shows representative cells that were stained for detection and localization of USP14 protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415) at a 1:300 dilution. Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane and cytoplasmic localization of USP14. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
