49-1023

antibody from Invitrogen Antibodies

Targeting: H4C14 H4/n, H4F2, H4FN, HIST2H4, HIST2H4A

Western blot (Supportive data in Antibodypedia)

ELISA (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

49-1023

Provider

Invitrogen Antibodies

Product name

H4K20me1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

Conc. Not Determined

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Histone extracts of HeLa cells (15 µg) were analyzed by western blot using the anti-H4K20me1 crude serum (Product # 49-1023), diluted 1:750 in TBS-Tween containing 5% milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

ELISA

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

To determine the titer, an ELISA was performed using the anti-H4K20me1 crude serum (Product # 49-1023). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the crude serum was estimated to be 1:8, 00.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP assays were performed using human osteosarcoma (U-2OS) cells, the anti-H4K20me1 crude serum (Product # 49-1023), and optimized PCR primer sets for qPCR. Each ChIP assay used sheared chromatin from 1.6 million cells and a volume of anti-H4K20me1 crude serum (2, 5, 10, or 15 µl). IgG (5 µg⁄IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. This figure shows the recovery, expressed as a percentage of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

A dot blot analysis was performed to test the cross reactivity of the anti-H4K20me1 crude serum (Product # 49-1023) with other modifications of histone H4 and with the unmodified protein. Other histone modifications include mono- and dimethylation of the same lysine and acetylation of the nearby lysine 16. To determine the cross reactivity, 0.2 to 100 pmol of peptide containing the respective histone modification was spotted on a membrane. Three different peptides for H4K16ac were used. The crude serum was used at a dilution of 1:20,000. At this dilution, the limit of detection of the antibody is 0.2 pmol. This figure shows a high specificity for the modification of interest.