702664

antibody from Invitrogen Antibodies

Targeting: FOXM1 FKHL16, HFH-11, HNF-3, INS-1, MPHOSPH2, MPP2, TGT3, trident

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

702664

Provider

Invitrogen Antibodies

Product name

FOXM1 Recombinant Rabbit Monoclonal Antibody (1H24L2)

Antibody type

Monoclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

1H24L2

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 Nuclear extracts (30 µg lysate) of MCF-7 (Lane 1), NTERA-2 cl.D1 (Lane 2), U-2 OS (Lane 3), A549 (Lane 4) and A-431 (Lane 5). The blots were probed with Anti-FOXM1 Recombinant Rabbit Monoclonal Antibody (Product # 702664, 1 µg/mL). A 84 kDa band corresponding to FOXM1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). 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 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, HCT 116 cells were fixed and permeabilized for detection of endogenous FOXM1 using Anti- FOXM1 Recombinant Rabbit Monoclonal Antibody (Product # 702664, 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 FOXM1 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating Nuclear localization of FOXM1. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of endogenous FOXM1 was performed on U-2 OS cells. Cells were either untreated (asynchronous) or treated with 100 ng/mL nocodazole for 16 hours to arrest cells in the G2/M phase of the cell cycle, where FOXM1 levels are known to be highest. Cells were then released from arrest with medium containing 10% FBS for 3 and 18 hours. FOXM1 was detected using Anti-FOXM1 Recombinant Rabbit Monoclonal Antibody (Product # 702664, 5 µg/10^6 cells) followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). Maximal expression of FOXM1 (panels a-d) was found to correlate with cells arrested in the G2/M phase as observed by staining with propidium iodide (panels e-h). Rabbit IgG was used as the isotype control to determine nonspecific binding. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770).

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of endogenous FOXM1 protein at specific gene loci using Anti-FOXM1 Recombinant Rabbit Monoclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-FOXM1 Recombinant Rabbit Monoclonal Antibody (Product # 702664, 5 µg) on sheared chromatin from 2 million HeLa cells using the MAGnify ChIP system kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoters of the CCNB1, CCND2 regions used as positive control target gene, and the region of the SAT2 satellite repeat, SAT Alpha used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.