- PA5-66995 - Invitrogen Antibodies COIL antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Coilin in human cell line RT-4 and human cell line U-251 MG. Samples were probed using a Coilin Polyclonal Antibody (Product # PA5-66995).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Coilin was achieved by transfecting HeLa with Coilin specific siRNAs (Silencer® select Product # s15663, s15662). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Coilin knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Coilin Polyclonal Antibody (Product # PA5-66995, 1:250 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 Coilin. Additional uncharacterized bands were also observed across the samples tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Coilin Polyclonal Antibody(Product # PA5-66995) and a 63kDa band corresponding to Coilin was observed across the cell lines tested. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), A549 (Lane 3), K-562 (Lane 4), Reh (Lane 5), MCF7 (Lane 6) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). 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 (1:250 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). Additional uncharacterized bands were also observed across the samples tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent staining of Coilin in human cell line SH-SY5Y shows localization to nucleus and nuclear bodies. Samples were probed using a Coilin Polyclonal Antibody (Product # PA5-66995).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Coilin 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 45 minutes at room temperature. The cells were labeled with Coilin Polyclonal Antibody (Product # PA5-66995) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (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 cajal bodies localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-66995