- 710788 - Invitrogen Antibodies POU5F1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane 1). The blots were probed with Anti-OCT4 Recombinant Rabbit Polyclonal Antibody (Product # 710788, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 39 kDa band corresponding to OCT4 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of OCT4 was achieved by transfecting NTERA-2 cells with OCT4 specific siRNA (Silencer® select Cat # s10871, Product # s10873). Western blot analysis (Fig a) was performed using Whole cell extract from the OCT4 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-OCT4 Recombinant Rabbit Polyclonal Antibody (Product # 710788, 2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to OCT4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, NTERA-2 cells were fixed and permeabilized for detection of endogenous OCT4 using Anti-OCT4 Recombinant Rabbit Polyclonal Antibody (Product # 710788, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of OCT4 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of OCT4. 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)
Main image
Experimental details: Enrichment of endogenous OCT4 protein at specific gene loci using Anti-OCT4 Recombinant Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-OCT4 Recombinant Rabbit Polyclonal Antibody (Product # 710788, 5 µg) on sheared chromatin from 2 million NTERA2 cells using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoters of the active SOX4, FOXA2, TNC region used as positive control target genes, and the region of the inactive MYOD, SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

710788