- TA347212 - OriGene H4-16 antibody

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Supportive validation

Submitted by: OriGene (provider)
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Experimental details: WB was performed on whole cell (25 ug, lane 1) and histone extracts (15 ug, lane 2 ) from HeLa cells, and on 1 ug of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the antibody against H4K5,8,12ac. The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.
Validation comment: WB

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H4K5,8,12ac in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:14,500.
Validation comment: ELISA

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: HeLa cells were fixed with 4% formaldehyde and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. Image shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. B, C, D and E: staining of the cells with the H4K5,8,12ac antibody after incubation of the antibody with 10 ng/ul of the following blocking peptides: H4K5,8,12 unmodified (B), H4K5,8,12ac (C), H2A.ZK5,7,11ac (D) and H4K5,8,12,16ac (E).
Validation comment: IF

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: ChIP was performed with the "iDeal ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Validation comment: Assay

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: ChIP was performed with 0.5 ug of the ab on sheared chromatin from 100,000 K562 cells. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Image shows the signal distribution along the complete length of chromosome 2 and a zoomin to a 600 kb region (B). C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls.
Validation comment: Assay

Supportive validation

Submitted by: OriGene (provider)
Main image
Experimental details: A Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Validation comment: DB

TA347212