- MA1-23246 - Invitrogen Antibodies COPS5 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of JAB1 was achieved by transfecting SH-SY5Y cells with JAB1 specific siRNAs (Silencer® select Product # s21627, s21628). Western blot analysis (Fig. a) was performed using whole cell extracts from the SH-SY5Y knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with JAB1 Monoclonal Antibody (6C3.38) (Product # MA1-23246, 0.5 µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ 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 JAB1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-JAB1 Monoclonal Antibody (Product # MA1-23246) and a ~37 kDa band corresponding to JAB1 was observed across the cell lines and tissues tested along with the uncharacterized bands (*) at ~80 kDa and ~30 kDa in the cell lines and tissues respectively. Whole cell extracts (30 ug lysate) of A-431 (Lane 1), A549 (Lane 2), HEK-293 (Lane 3), HeLa (Lane 4), MCF-7 (Lane 5), HT-29 (Lane 6), SW-480 (Lane 7), HCT 116 (Lane 8), Mouse Lung (Lane 9), Rat Lung (Lane 10) and Mouse spleen (Lane 11) 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 (1:2000 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of JAB1 was performed using 70% confluent log phase HeLa 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 JAB1 Monoclonal Antibody (Product # MA1-23246) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) 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 nuclear and 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)
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Experimental details: Immunohistochemical analysis using JAB1 Monoclonal Antibody (6C3.38) (Product # MA1-23246).

MA1-23246