- PA5-17441 - Invitrogen Antibodies CBX5 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HP1-alpha in lysates from HeLa and NIH/3T3 cells using HP1-alpha polyclonal antibody (Product # PA5-17441).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Chromobox protein homolog 5 was achieved by transfecting HeLa with Chromobox protein homolog 5 specific siRNAs (Silencer® select Product # s23884, s23883). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Chromobox protein homolog 5 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HP1 alpha Polyclonal Antibody (Product # PA5-17441, 1:1000 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 Chromobox protein homolog 5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HP1 alpha Polyclonal Antibody (Product # PA5-17441) and a 25 kDa band corresponding to Chromobox protein homolog 5 was observed across tested cell lines. Nuclear enriched extracts (40 µg lysate) of MOLT-4 (Lane 1), HeLa (Lane 2), SH-SY5Y (Lane 3), PC-3 (Lane 4), Hep G2 (Lane 5), NIH/3T3 (Lane 6) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). 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-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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Chromobox protein homolog 5 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with HP1 alpha Polyclonal Antibody (Product # PA5-17441) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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 predominantly nuclear 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 of HP1-alpha in paraffin-embedded human breast carcinoma using a HP1-alpha polyclonal antibody (Product # PA5-17441) in the presence of control peptide (left) or HP1 alpha blocking peptide (right).

PA5-17441