MA1-12420

antibody from Invitrogen Antibodies

Targeting: LEF1 TCF10, TCF1ALPHA, TCF7L3

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

MA1-12420

Provider

Invitrogen Antibodies

Product name

LEF1 Monoclonal Antibody (REMB6)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

A suggested positive control for this product is Jurkat cell lysate.

Reactivity

Human, Mouse

Host

Mouse

Isotype

IgG

Antibody clone number

REMB6

Vial size

250 µg

Concentration

1 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

References

Submitted references

Association of nuclear-localized Nemo-like kinase with heat-shock protein 27 inhibits apoptosis in human breast cancer cells.

Shaw-Hallgren G, Chmielarska Masoumi K, Zarrizi R, Hellman U, Karlsson P, Helou K, Massoumi R
PloS one 2014;9(5):e96506

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of LEF1 using a monoclonal antibody (Product # MA1-12420).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-LEF1 Monoclonal Antibody (REMB6) (Product # MA1-12421) and a 55 kDa band corresponding to LEF1 was observed across the panel tested except Caco-2 and HeLa which are reported to be negative. Fresh Nuclear enriched extracts (40 µg lysate) of MOLT-4 (Lane 1), Jurkat (Lane 2), Caco-2 (Lane 3), HeLa (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (2.5 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band at ~150 kDa was also observed in all the cell lines tested.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of LEF1 was performed using 70% confluent log phase MOLT-4 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 LEF1 Monoclonal Antibody (REMB6) (Product # MA1-12421) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:3000 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). Panel d represents the merged image showing nucleus localization. Panel e represents merged image for Caco-2 cells showing no staining for LEF-1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin Immunoprecipitation (ChIP) assay of endogenous LEF1 protein using LEF1 Antibody: ChIP was performed using LEF1 Monoclonal Antibody (REMB6) (Product # MA1-12421, 5 µg) on sheared chromatin from HCT 116 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Mouse IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to OLIG1, AXIN2, c myc promoter (region1- PR1), c myc promoter (region2- PR2) and MSX1 (active) and SAT alpha, SAT2 satellite repeats and Actin beta (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.