- PA1-23693 - Invitrogen Antibodies FN1 antibody

Sari AR, Rufaut NW, Jones LN, Sinclair RD
International journal of trichology 2016 Jul-Sep;8(3):121-9

Zobel K, Hansen U, Galla HJ
Cell and tissue research 2016 Aug;365(2):233-45

Esen N, Wagoner G, Philips N
Journal of neuroimmunology 2010 Jan 25;218(1-2):83-93

Chade AR, Zhu XY, Grande JP, Krier JD, Lerman A, Lerman LO
Journal of hypertension 2008 Aug;26(8):1651-60

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of FN1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR616644_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of FN1 was performed by loading 30 µg of HepG2 Wild Type (Lane 1), HepG2 Cas9 (Lane 2) andHepG2 FN1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Fibronectin Polyclonal Antibody (Product # PA1-23693, 2 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to FN1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Fibronectin Polyclonal Antibody (Product # PA1-23693) and a ~260 kDa band corresponding to Fibronectin was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Hep G2 treated with 1X PTI for 4h (Lane 2), Hep G2 treated with 10 µm FLI-06 for 4h (Lane 3), HEL 92.1.7 (Lane 4), HEL 92.1.7 treated with 1X PTI for 4h (Lane 5), HEL 92.1.7 treated with 10 µm FLI-06 for 4h (Lane 6) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). The blot was probed with the primary antibody (2 µg/mL) 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). Anti-Fibronectin Polyclonal Antibody (Product # PA1-23693) showed enhanced pick up in positive cell line Hep G2 upon treatment with secretion blockers as compared to in negative cell line HEL 92.1.7 treated with the same blockers.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Fibronectin was performed using 70% confluent log phase Hep G2 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 45 minutes at room temperature. The cells were labeled with Fibronectin Monoclonal Antibody (3F12) (Product # MA5-14737) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), 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 showing cytosolic localization. Panel e represents untreated Hep G2 cells with no expression. Panel f represents control cells with no primary antibody to assess background The images were captured at 60X magnification. Cells treated with 1X PTI for 4h (Panel a) showed enhanced expression as compared to untreated cells (Panel e).

PA1-23693