- MIF2601 - Invitrogen Antibodies FN1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Fibronectin was performed by loading 30 µg of the indicated whole cell lysates and 10 µL PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using the G2 Fast Blotter (Product # 62288) and blocked with 5% Milk/TBST for at least 1 hour at room temperature. Fibronectin was detected at ~262 kDa using a Fibronectin mouse monoclonal antibody (Product # MIF2601) at a concentration of 2 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a Superclonal goat anti-mouse IgG-HRP secondary antibody (Product # A28177) at a dilution of 1:2,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Fibronectin was performed by loading 25 µg of human plasma lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a Fibronectin monoclonal antibody (Product # MIF2601) at a dilution of 1:300 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG + IgM (H+L) cross-adsorbed secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~263 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Fibronectin 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 Fibronectin was performed by loading 30 µg of Hep G2 Wild Type (Lane 1), Hep G2 Wild Type treated with 1X PTI for 4hrs (Lane 2), Hep G2 Cas9 (Lane 3), Hep G2 Cas9 treated with 1X PTI for 4hrs (Lane 4), Hep G2 Fibronectin KO (Lane 5) and Hep G2 Fibronectin KO treated with 1X PTI for 4hrs (Lane 6) whole cell extracts. The samples 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 Anti-Fibronectin Monoclonal Antibody (3F12) (Product # MIF2601, 2 µg/mL dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 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 Fibronectin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Direct ELISA analysis of Ferritin was performed by coating wells of a 96-well plate with 100 µL per well of Human Ferritin or BSA diluted in carbonate/bicarbonate buffer (Product # 28382), starting at a concentration of 1 µg/mL and serially diluting 2-fold to a concentration of 2 ng/mL, overnight at 4C. Wells of the plate were washed, blocked with StartingBlock blocking buffer (Product # 37538), and incubated with 100 µL per well of a mouse anti-ferritin monoclonal antibody (Product # MIF2601) at a concentration of 1 µg/mL for 1 hour at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:25000 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 10 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid, and absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Fibronectin (green) in HepG2 cells. The cells were fixed with formalin for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 5% Normal Goat Serum (Product # 31872) for 15 minutes at room temperature. Cells were stained with or without Fibronectin monoclonal antibody (Product # MIF2601), at a dilution of 1:100 overnight at 4C, and then incubated with a DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35503) at a dilution of 1:1000 for 30 minutes at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Fibronectin Mouse Monoclonal Antibody (Product # MIF2601) at 5 µg/mL in 0.1% BSA and incubated for overnight at 4 degree Celsius and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000) for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Golgi complex localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Fibronectin was achieved by transfecting Hep G2 cells with Fibronectin specific siRNA (Silencer® select Cat # s5322). Immunofluorescence analysis was performed on Hep G2 cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with Fibronectin specific siRNA (panel c,f) Cells were fixed, permeabilized, and labelled with Fibronectin Mouse monoclonal Antibody (Product # MIF2601, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of specific signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to Fibronectin(green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Sandwich ELISA of Fibronectin was performed by coating wells of a 96-well plate with 100 µL of an fibronectin chicken polyclonal antibody (Product # PA1-27507) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 100 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 28382) for 2 hours, and 100 µL of recombinant human plasma fibronectin protein ranging from 1.6-1000 ng/mL were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of a fibronectin mouse monoclonal antibody (Product # MIF2601) diluted to a concentration of 1 µg/mL was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a Streptavidin-HRP (Product # 21126) was incubated for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Direct ELISA analysis of Ferritin was performed by coating wells of a 96-well plate with 100 µL per well of Human Ferritin or BSA diluted in carbonate/bicarbonate buffer (Product # 28382), starting at a concentration of 1 µg/mL and serially diluting 2-fold to a concentration of 2 ng/mL, overnight at 4C. Wells of the plate were washed, blocked with StartingBlock blocking buffer (Product # 37538), and incubated with 100 µL per well of a mouse anti-ferritin monoclonal antibody (Product # MIF2601) at a concentration of 1 µg/mL for 1 hour at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:25000 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 10 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid, and absorbances were read on a spectrophotometer at 450-550 nm.

MIF2601

Antibody data