- MA5-24606 - Invitrogen Antibodies REST antibody

Submitted references Soluble guanylate cyclase signalling mediates etoposide resistance in progressing small cell lung cancer.

Schenk MW, Humphrey S, Hossain ASMM, Revill M, Pearsall S, Lallo A, Brown S, Bratt S, Galvin M, Descamps T, Zhou C, Pearce SP, Priest L, Greenhalgh M, Chaturvedi A, Kerr A, Blackhall F, Dive C, Frese KK
Nature communications 2021 Nov 17;12(1):6652

The Rare YAP1 Subtype of SCLC Revisited in a Biobank of 39 Circulating Tumor Cell Patient Derived Explant Models: A Brief Report.

Pearsall SM, Humphrey S, Revill M, Morgan D, Frese KK, Galvin M, Kerr A, Carter M, Priest L, Blackhall F, Simpson KL, Dive C
Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2020 Dec;15(12):1836-1843

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-REST Monoclonal Antibody (CL0381) (Product # MA5-24606) and a 200 kDa band corresponding to REST was observed across cell lines tested. Nuclear enriched extracts (60 µg lysate) of HeLa (Lane 1), T-47D (Lane 2) and HCT 116 (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1.4 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of REST was achieved by transfecting HeLa with REST specific siRNAs (Silencer® select Product # s11932 and s11933). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the REST knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with REST Monoclonal Antibody (CL0381) (Product # MA5-24606, 1.4 µg/mL ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to REST. An uncharacterized band (*) was observed at around 65 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of REST in U-2 OS cells using REST Monoclonal Antibody (CL0381) (Product # MA5-24606), showing specific staining in the nucleoplasm in green. Microtubule- and nuclear probes are visualized in red and blue, respectively (where available).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of REST was performed using 70% confluent log phase HeLa 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 1 hour at room temperature. The cells were labeled with REST Monoclonal Antibody (CL0381) (Product # MA5-24606) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (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). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of REST was performed using 70% confluent log phase NTERA-2 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 REST Monoclonal Antibody (CL0381) (Product # MA5-24606) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 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). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical staining of REST in human testis using REST Monoclonal Antibody (CL0381) (Product # MA5-24606) shows moderate to strong nuclear positivity in cells in seminiferous ducts.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical staining of REST in human tonsil using REST Monoclonal Antibody (CL0381) (Product # MA5-24606) shows moderate to strong nuclear positivity in lymphoid cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical staining of REST in human endometrium using REST Monoclonal Antibody (CL0381) (Product # MA5-24606) shows moderate nuclear positivity in glandular and stromal cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical staining of REST in human cerebral cortex using REST Monoclonal Antibody (CL0381) (Product # MA5-24606) shows no nuclear positivity in neuronal cells as expected.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous REST was performed using REST Monoclonal Antibody (CL0381) (Product # MA5-24606, 5 µg) on sheared chromatin from NTERA-2 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 MEIS1, MEIS2, HOXD11, TBX3, DLX5 (all active loci) and SAT alpha (inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-24606

Antibody data