702443

antibody from Invitrogen Antibodies

Targeting: SUV39H1 KMT1A, SUV39H

Antibody data

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Validation data

SUV39H1 protein structure - 702443 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SUV39H1 was achieved by transfecting HeLa cells with SUV39H1 specific siRNA (Silencer® select Cat # s13658 and s13659). Western blot analysis (Fig a) was performed using Whole cell extract from the SUV39H1 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-SUV39H1 Recombinant Rabbit Monoclonal Antibody (Product # 702443, 1-3 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 SUV39H1.

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 T98G (Lane 1), HCT 116 (Lane 2), COS-7 (Lane 3), A-431 (Lane 4), HeLa (Lane 5), Hep G2 (Lane 6), A549 (Lane 7) and HEK-293 (Lane 8). The blots were probed with Anti-SUV39H1 Recombinant Rabbit Monoclonal Antibody (Product # 702443, 0.5 µg/mL). A 48 kDa band corresponding to SUV39H1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). 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: Mouse livers have been minced, treated with ACK solution (removing red blood cells) and lysed in RIPA buffer (1% SDS). The samples were incubated 2h on ice, vortexing every 15min. The samples were quickly sonicated for 2min, spun down at 15,000rpm for 15min at 4C and supernatant was collected and used to quantify total protein extract via Bradford assay. 10ug total protein extract were loaded on 4-12% SDS gel. Lane 1 is wildtype SUV39H1 and lane 2 is mutant SUV39H1. After blocking membrane in milk, membrane was incubated with primary monoclonal SUV39H1 antibody (Product # 702443) at a dilution of 1:1000 overnight at 4 deg. Secondary antibody was used at a dilution of 1:5000. SuperSignal West Pico Chemiluminescent substrate (Product # 34577) was used for detection. Data courtesy of the Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, U2OS cells were fixed and permeabilized for detection of endogenous SUV39H1 using Anti- SUV39H1 Recombinant Rabbit Monoclonal Antibody (Product # 702443, 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 SUV39H1 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of panels a, b and c clearly demonstrating nuclear localization of SUV39H1. 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: Localization of endogenous SUV39H1 with heterochromatin was observed by immunofluorescence microscopy using Anti- SUV39H1 Recombinant Rabbit Monoclonal Antibody. NIH3T3 mouse fibroblast cells were stained with Anti- SUV39H1 Recombinant Rabbit Monoclonal Antibody (Product # 702443, 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 detection and localization of SUV39H1 protein in the cell nucleus (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) is a composite image of panels a and b clearly demonstrating localization of SUV39H1 with heterochromatin. Arrows indicate regions of high chromatin density, identified as heterochromatin, as determined by increased uptake of DAPI. The images were captured at 100X magnification.