- PA5-65088 - Invitrogen Antibodies MCM4 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MCM4 in human cell line RT-4, human cell line U-251 MG, human plasma, human liver tissue and human tonsil tissue. Samples were probed using a MCM4 Polyclonal Antibody (Product # PA5-65088).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of MCM4 was achieved by transfecting SH-SY5Y with MCM4 specific siRNAs (Silencer® select Product # S8592, S8593). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the MCM4 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MCM4 Polyclonal Antibody (Product # PA5-65088, 1:200 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5000 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 MCM4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-MCM4 Polyclonal Antibody (Product # PA5-65088) and a 110 kDa band corresponding to MCM4 was observed across cell lines and tissues tested. Nuclear enriched extracts (30 µg lysate) of Neuro-2a (Lane 1), NIH/3T3 (Lane 2), NIH:OVCAR-3 (Lane 3), U-2 OS (Lane 4), SH-SY5Y (Lane 5), Mouse testis (Lane 6), Mouse Thymus (Lane 7), Mouse lung (Lane 8) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (1:200 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:5000 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)
Main image
Experimental details: Immunofluorescent staining of MCM4 in human cell line U-2 OS shows localization to nucleoplasm. Samples were probed using a MCM4 Polyclonal Antibody (Product # PA5-65088).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MCM4 was performed using 70% confluent log phase SH-SY5Y 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 MCM4 Polyclonal Antibody (Product # PA5-65088) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (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 of SH-SY5Y cells showing nuclear localization for MCM4. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-65088