- 701682 - Invitrogen Antibodies SMAD4 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of SMAD4 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR1106592_LV and CRISPR1106595_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of SMAD4 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa SMAD4 KO (Lane 3) whole cell extracts. The blot was probed with Anti-SMAD4 Recombinant Rabbit Monoclonal Antibody (10H10L15) (Product # 701682) using 1 µg/mL dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to SMAD4. An uncharacterized band was observed at ~40 kDa.

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 A549 (Lane 1), A549 serum starved for 4 hours (Lane 2), PC -3 (Lane 3), PC-3 serum starved for 4 hours (Lane 4).The blots were probed with Smad4 Recombinant Rabbit Monoclonal Antibody (Product # 701682 0.5-2 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 clear 60 kDa band corresponding to Smad4 was observed across 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%skimmedmilk. 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: Western blot was performed using Anti-SMAD4 Recombinant Rabbit Monoclonal Antibody (10H10L15) (Product # 701682) and a 60 kDa band corresponding to Mothers against decapentaplegic homolog 4, along with an uncharacterized band (*) was observed across positive cell line (HCT 116); and not across negative cell lines (HT-29 and SW480). The same showed an increased expression in HCT 116 upon serum starvation for 4 hours. Whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), serum starved HCT 116 (Lane 2), HT-29 (Lane 3) and SW480 (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). 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 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) 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 analysis of Mothers against decapentaplegic homolog 4 was performed using 70% confluent log phase HCT 116 (Positive) and SW480 (Negative) 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 SMAD4 Recombinant Rabbit Monoclonal Antibody (10H10L15) (Product # 701682) at 2 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) (1:2000 dilution), 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 dilution). Panel d represents the merged image showing nucleus and cytoplasm localization in HCT 116 cells, against Panel e representing SW480 which shows no signal. Panel f 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: Immunofluorescence was performed on fixed and permeabilized MCF7 cells for detection of Smad4 using Anti-Smad4 Recombinant Rabbit Monoclonal Antibody (Product # 701682, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of Smad4 (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic and nuclear localization of Smad4. 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 Smad4 was performed on A549 cells labeled with ABfinityªSmad4 Recombinant Rabbit Monoclonal Antibody (Product# 701682, 2-4 ug/ 1M cells) or with rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonalª 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 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer (4468770).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of SMAD4 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of recombinant monoclonal SMAD4 antibody (Product # 701682) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of SMAD4 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of recombinant monoclonal SMAD4 antibody (Product # 701682) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

701682

Antibody data