- PA1-41120 - Invitrogen Antibodies HDAC1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HDAC1 in 293 cell lysate probed with a HDAC1 polyclonal antibody (Product # PA1-41120).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HDAC1 in HEK293 cell lysate. Sample was incubated in HDAC1 polyclonal antibody (Product # PA1-41120).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HDAC1 in 0.5 mg/mL Hek293 lysate. Samples were incubated in HDAC1 polyclonal antibody (Product # PA1-41120). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of HDAC1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1087187_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HDAC1 was performed by loading 30 µg of HeLa Wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa HDAC1 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HDAC1 Polyclonal Antibody (Product # PA1-41120, 2 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to HDAC1. Uncharacterized band was observed in all the samples at 51 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HDAC1 was achieved by transfecting MCF7 cells with HDAC1 specific siRNAs (Silencer® select Product # s73). Western blot analysis (Fig. a) was performed using Modified whole cell extracts (1% SDS) from the HDAC1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-HDAC1 Polyclonal Antibody (Product # PA1-41120, 2µg/ml) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). 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 HDAC1..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HDAC1 Polyclonal Antibody (Product # PA1-41120) and ~55kDa band corresponding to HDAC1 was observed in MCF-7, HeLa, HEK-293 and NIH/3T3 cells. Modified whole cell extracts (1% SDS) (30 ug lysate) of MCF-7 (Lane 1), HeLa (Lane 2), HEK-293 (Lane 3) and NIH/3T3 (Lane 4) were electrophoresed using NuPAGE® 4-12% Bis-Tris gel (Product # NP0322BOX). 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 (2µg/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 HDAC1 was performed using 70% confluent log phase MCF-7 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 HDAC1 Polyclonal Antibody (Product # PA1-41120) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). 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)
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Experimental details: Immunohistochemical analysis of HDAC1 in Human testis. Samples were incubated in HDAC1 polyclonal antibody (Product # PA1-41120) using a dilution of 1:250. Bond Rx autostainer (Leica Biosystems). The assay involved 20 minutes of heat induced antigen retrieval (HIER) using 10mM sodium citrate buffer (pH 6.0) and endogenous peroxidase quenching with peroxide block. The sections were incubated with primary antibody for 30 minutes and Bond Polymer Refine Detection (Leica Biosystems) with DAB was used for signal development followed by counterstaining with hematoxylin. Whole slide scanning and capturing of representative images was performed using Aperio AT2 (Leica Biosystems). Staining was performed by Histowiz.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous HDAC1 protein using HDAC1 Polyclonal Antibody: ChIP was performed using HDAC1 Polyclonal Antibody (Product # PA1-41120, 5 µg) on sheared chromatin from MCF-7 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CDKN1-PR, GAPDH- TSS and FOSL1-PR as active binding regions and FOSL1- Exon1 and SAT alpha as inactive binding regions. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method. PR: Promoter, TSS: Transcription start site.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous HDAC1 protein using Anti-HDAC1 Antibody: ChIP was performed using Anti-HDAC1 Rabbit Polyclonal Antibody (Product # PA1-41120, 5 µg) on sheared chromatin from HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CDKN1A Intron 1, transcriptional start site and gene body (+2kb) of GAPDH, RPL30 exon 5 and SATA satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA1-41120