- 49-1025 - Invitrogen Antibodies HDAC1 antibody

Submitted references The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer.

Mao X, Gauche C, Coughtrie MW, Bui C, Gulberti S, Merhi-Soussi F, Ramalanjaona N, Bertin-Jung I, Diot A, Dumas D, De Freitas Caires N, Thompson AM, Bourdon JC, Ouzzine M, Fournel-Gigleux S
Oncogene 2016 Sep 22;35(38):5043-55

Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs.

Plotkin A, Volmar CH, Wahlestedt C, Ayad N, El-Ashry D
Breast cancer research and treatment 2014 Sep;147(2):249-63

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Whole cell extracts (25 μg, lane 1) and nuclear extracts (25 μg, lane 2) from HeLa cells were analysed by Western blot using the anti-HDAC1 antibody (Product # 49-1025) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right (expected size: 55 kDa); the marker (in kDa) is shown on the left.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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 # 49-1025, 1:1000 dilution) 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)
Main image
Experimental details: Western blot was performed using Anti-HDAC1 Polyclonal Antibody (Product # 49-1025) and ~55 kDa band corresponding to HDAC1 was observed in MCF-7, HeLa, HEK-293, NIH/3T3, PC-12 cells and Mouse brain. Modified whole cell extracts (1% SDS) (30 µg lysate) of MCF-7 (Lane 1), HeLa (Lane 2), HEK-293 (Lane 3), NIH/3T3 (Lane 4), PC-12 (Lane 5) and Mouse brain (Lane 6) 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 (1:1000 dilution) 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)
Main image
Experimental details: To determine the titer, an ELISA was performed using anti-HDAC1 crude serum, purified anti-HDAC1 antibodies (Product # 49-1025), and the column flow through obtained from the antibody purification step. The antigen used was the C-terminal peptide used for immunization. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:4, 250.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: To determine the titer of the antibody, an ELISA was performed using a serial dilution of anti-HDAC1 antibody (Product # 49-1025), crude serum and flow through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:75,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Mouse differentiated ES cells were formaldehyde fixed, permeabilized with Triton® X-100 (Dow Chemical Co) and then blocked with PBS containing BSA. Cells were labeled with the anti-HDAC1 (diluted 1:200 and incubated for 45 minutes at room temperature) followed by labeled goat anti-rabbit secondary antibody. Subsequently, RNA FISH (fluorescence in situ hybridization) was performed to detect Xist RNA (green signal). Nuclei were DAPI-stained to specifically label the DNA. As expected, both DAPI and HDAC1 staining occur in the nucleus of the cells. Note the broadly dispersed characteristic pattern of this protein mark in interphase chromatin but its exclusion from constitutive heterochromatin in mouse cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were stained with the anti-HDAC1 antibody (Product # 49-1025) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the HDAC1 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP assays were performed using U-2OS cells, the anti-HDAC1 antibody (Product # 49-1025), and optimized PCR primer sets for qPCR. Each ChIP assay used sheared chromatin from 1 million cells and 2.4 µg of anti-HDAC1 antibody. The results, expressed as the relative amount of immunoprecipitated DNA compared to input DNA (% of input) are shown. Left: ChIP results using the anti-HDAC1 antibody (bar 2) or beads only (bar 1) and PCR primers specific for p21. The occupancy of p21 (a known HDAC1 target gene) by HDAC1 is clearly demonstrated. Right: ChIP results using the anti-HDAC1 antibody (bar 4) or beads only (bar 3) and PCR primers for GAPDH (used as negative control).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 2 µg of the anti-HDAC1 antibody (Product # 49-1025) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. The figure shows the peak distribution along the complete sequence and a 1 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP was performed with the anti-HDAC1 antibody (Product # 49-1025) on sheared chromatin from 4,000,000 HeLa cells. An antibody titration consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers specific for the EIF4A2 and GAPDH promoters, used as positive controls, and for the MYOD1 gene and Sat2 satellite repeat, used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 2 µg of the anti-HDAC1 antibody (Product # 49-1025) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. The figure shows the peak distribution along the complete sequence and a 1 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D).

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 # 49-1025, 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.

49-1025

Antibody data