- NBP2-59186 - Novus Biologicals H3C14 antibody

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Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Western Blot: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - Western blot was performed using histone extracts from HeLa cells (HeLa HE, 15 ug) and the antibody against H3K4me3 diluted 1:500 in TBS-Tween containing 5% skimmed milk. A molecular weight marker (in kDa) is shown on the left, the position of the protein of interest is shown on the right.

Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Western Blot: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - Western blot was performed on whole cell (40 ug, lane 1) and histone extracts (15 ug, lane 2) from HeLa cells, and on 1 ug of recombinant histone H2A, H2B, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the antibody against H3K4me3. The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. Observed molecular weight is ~17 kDa.

Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Chromatin Immunoprecipitation: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - ChIP assays were performed using human HeLa cells, the antibody against H3K4me3 and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 ug of antibody per ChIP experiment was analyzed. IgG (2 ug/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes

Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Chromatin Immunoprecipitation: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - ChIP assays were performed using human HeLa cells, the Ab against H3K4me3 and PCR primers for qPCR. ChIP was performed using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). IgG (2 ug/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene, used as negative control. The graph shows the recovery, expressed as a % of input (the relative amount of IP DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes.

Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Chromatin Immunoprecipitation: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3 modifications and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me3 modification. At higher concentrations some H3K4me2 is also precipitated.

Supportive validation

Submitted by: Novus Biologicals (provider)
Main image
Experimental details: Dot Blot: Histone H3 [Trimethyl Lys4] Antibody [NBP2-59186] - A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K4me3 with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. Figure shows a high specificity of the antibody for the modification of interest.

NBP2-59186