720333

antibody from Invitrogen Antibodies

Targeting: SMAD9 MADH6, MADH9, SMAD8, SMAD8/9

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

720333

Provider

Invitrogen Antibodies

Product name

SMAD9 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), A549 (Lane 2), HEK-293 (Lane 3) and HeLa (Lane 4). The blots were probed with Anti-Smad9 Rabbit Polyclonal Antibody (Product # 720333, 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 53 kDa band corresponding to Smad9 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous SMAD9 using Anti- SMAD9 Rabbit Polyclonal Antibody (Product # 720333, 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 SMAD9 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 nuclear localization of SMAD9. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.