- TA347214 - 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 H4K5ac. The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. 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 H4K5ac 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 1:8,900.
Validation comment: ELISA

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: ChIP was performed with 1 ug of the ab on sheared chromatin from 1 million HeLa cells. 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 1 and a zoomin to a 500 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. The position of the amplicon used for validating the QPCR results is shown with arrow.
Validation comment: Assay

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: ChIP assays using HeLa cells on sheared chromatin from 1 million cells. Titration of 1,2,5&10ug antibody per ChIP was analysed. IgG (2 ug/IP) was used as negative control. qPCR primers were for promoter of active gene EIF4A2 and for a region 1 kb upstream of GAPDH as positive controls, and for inactive MYOD1 gene and the Sat2 satellite repeat region as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).
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 1:5,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Validation comment: DB

TA347214