- TA347213 - OriGene H4-16 antibody

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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 H4K5,8,12,16ac 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 1:21,200.
Validation comment: ELISA

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: INIH3T3 cells were stained with the ab against H4K5,8,12,16ac 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 labeled with the H4K5,8,12,16ac 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.
Validation comment: IF

Supportive validation

Submitted by: OriGene (provider)
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Experimental details: ChIP was performed with 2 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 5 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. 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 (sheared chromatin from 1 million cells). Titration of 1, 2, 5 and 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 and the Sat2 satellite repeat region used 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)
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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

TA347213