- MA5-21203 - Invitrogen Antibodies CBX1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of CBX1 expressed in transfected 293T cell line by anti-CBX1 monoclonal antibody (Product # MA5-21203), clone 4E12. Lane 1: CBX1 transfected lysate (21.4 KDa). Lane 2: Non-transfected lysate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HP1 was achieved by transfecting MCF7 with HP1 specific siRNAs (Silencer® select Product # s21549). Western blot analysis (Fig. a) was performed using whole cell extracts from the HP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HP1 Monoclonal Antibody (Product # MA5-21203, 1ug/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 0.25µg/ml, 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 HP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HP1 beta Monoclonal Antibody (Product # MA5-21203) and a 25kDa band corresponding to HP1 was observed across the cell lines and tissues tested. Modified whole cell extracts (30ug) of HeLa (Lane 1), MCF7 (Lane 2), A-431 (Lane 3), U-2 OS (Lane 4), PC-3 (Lane 5), Raji (Lane 6), IMR32 (Lane 7), Hep G2 (Lane 8), Mouse Liver (Lane 9) and Rat Liver (Lane 10) 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 (1ug/ml) 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 HeLa cells using an anti-CBX1 monoclonal antibody (Product # MA5-21203). (antibody concentration 10 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of HP1 was performed using MCF7 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 HP1 beta Monoclonal Antibody (Product # MA5-21203) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 composite image showing nuclear localization of HP1. 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: Detection limit for recombinant GST tagged CBX1 using anti-CBX1 monoclonal antibody (Product # MA5-21203) is 0.1 ng/mL as a capture antibody.

MA5-21203