- 701980 - Invitrogen Antibodies NOTCH2 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) U-87 MG (Lane 1), Karpas 299 (Lane 2), MCF7 (Lane 3), A-431 (Lane 4), HEK-293 (Lane 5), SH-SY5Y (Lane 6). The blots were probed with Anti- Notch2 Recombinant Rabbit Monoclonal Antibody (Product # 701980, 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 ~110 kDa band corresponding to the transmembrane region of Notch2 and a ~265 kDa band corresponding to full-length protein of Notch2 was detected. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), 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 overnight wet transfer system. 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 NOTCH2 was achieved by transfecting HaCaT with NOTCH2 specific siRNAs (Silencer® select Product # S9638, S9639). Western blot analysis (Fig. a) was performed using Whole cell extracts from the NOTCH2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NOTCH2 Antibody (18H4L20), ABfinity Rabbit Monoclonal (Product # 701980, 1:250 ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 NOTCH2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NOTCH2 Antibody (18H4L20), ABfinity Rabbit Monoclonal (Product # 701980) and 300-350 kDa band corresponding to full length and 110 kDa band corresponding to cleaved fragment of NOTCH2 was observed across cell lines tested. Whole cell extracts (40 µg lysate) of HaCaT (Lane 1), MDA-MB-231 (Lane 2), U-87-MG (Lane 3), KARPAS 299 (Lane 4), SH-SY5Y (Lane 5), A-431 (Lane 6) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). 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 (1:250) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) 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 was performed on fixed and permeabilized U-87 MG cells for detection of endogenous Notch2 using Anti-Notch2 Recombinant Rabbit Monoclonal Antibody (Product # 701980, 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 Notch2 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 localization of Notch2 in the membrane and cytoplasm. Panel e) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of Notch2 was performed on U-87 MG cells labeled with ABfinity™ Anti-Notch2 Recombinant Rabbit Monoclonal Antibody (Product# 701980, 5 ug/ 1M cells) or with rabbit isotype control at 0.5 ug/ml and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).

701980