- 711280 - Invitrogen Antibodies LCN2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of A549 (Lane 1), A549 treated with Thapsigargin (500 nM for 48 hrs) (Lane 2), HT-29 (Lane 3) and HT-29 treated with Thapsigargin (500 nM for 48 hrs) (Lane 4). The blots were probed with Anti-Lipocalin 2 Recombinant Rabbit Polyclonal Antibody (Product # 711280, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 22 kDa band corresponding to Lipocalin 2 was observed across the 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)
Main image
Experimental details: Knockdown of Lipocalin 2 was achieved by transfecting PC-3 cells with Lipocalin 2 specific siRNA (Silencer® select Product # s8114+s8112). Western blot analysis (Fig a) was performed using whole cell extracts from Lipocalin 2 knockdown cells (Lane 2) and non-specific scrambled siRNA transfected cells (Lane 1). The blots were probed with Anti-Lipocalin 2 Recombinant Rabbit Polyclonal Antibody (Product # 711280, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 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 Lipocalin 2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of A549 (Lane 1), A549 treated with Thapsigargin (500 nM for 48 hrs) (Lane 2), HT-29 (Lane 3) and HT-29 treated with Thapsigargin (500 nM for 48 hrs) (Lane 4). The blots were probed with Anti-Lipocalin 2 Recombinant Rabbit Polyclonal Antibody (Product # 711280, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 22 kDa band corresponding to Lipocalin 2 was observed across the 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)
Main image
Experimental details: For immunofluorescence analysis, A549 cells were fixed and permeabilized for detection of endogenous Lipocalin2 using Anti-Lipocalin2 Recombinant Rabbit Polyclonal Antibody (Product # 711280, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) is stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300) Panel a-d) shows representative undifferentiated cells that were stained for detection and localization of Lipocalin2 protein (green) with no signal. Panel e-h) clearly demonstrate enhanced signal of Lipocalin2 in cytoplasm of cells treated with thapsigargin (500 nM 48hrs). The images were captured at 60X magnification.

711280