49-1024

antibody from Invitrogen Antibodies

Targeting: H4C1 H4FA, HIST1H4A

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

49-1024

Provider

Invitrogen Antibodies

Product name

Anti-Tri-Methyl-Histone H4 (Lys20) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

Lot Dependent

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Histone (acid) extracts of NB4 cells were analyzed by western blot using the anti-H4K20me3 crude serum (Product # 49-1024), diluted 1:750 in TBS-Tween containing 5% milk. The location of the protein of interest is indicated.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Histone (acid) extracts of NB4 cells were analysed by Western blot using the anti-H4K20me3 antibody (Product # 49-1024), diluted 1:750 in TBS-Tween containing 5% skimmed milk. The location of the protein of interest is indicated on the right.

ELISA

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

To determine the titer, an ELISA was performed using anti-H4K20me3 crude serum, purified anti-H4K20me3 antibodies, and the column flow through obtained from the antibody purification step. The antigen used was the peptide including the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the crude serum was estimated to be 1:700 for crude serum (Product # 49-1021) and 1:350 for affinity purified antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

To determine the titer, an ELISA was performed using a serial dilution of the anti-H4K20me3 antibody (Product # 49-1024). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:700.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP assays were performed using undifferentiated human teratocarcinoma cells (NCCIT), the anti-H4K20me3 crude serum (Product # 49-1024), and optimized PCR primer sets for PCR. Each ChIP assay used sheared chromatin from 10,000 cells and a volume of pre-immune serum (yellow bars) or crude serum (red and blue bars). H3K9me3 is a marker for heterochromatin. Therefore, we used the promoter of a house keeping gene c-fos, which is under active transcription, as negative PCR control. SAT-2, present in heterochromatin, is used as positive PCR control. In red: recovery (% of input) for the antiserum H4K20me3 (D87 = day 87 of the immunization program) used at dilution 1:500, using primer pairs for negative PCR control (bar 2) and positive PCR control (bar 5). In blue: recovery (% of input) for the antiserum H4K20me3 (D87) used at dilution 1:100, using primer pairs for negative PCR control (bar 3) and positive PCR control (bar 6). In purple: recovery (% of input) for the pre-immune serum (D0) using primer pairs for negative PCR control (bar 1) and positive PCR control (bar 4). The percentage of recovery represents the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis. The antibody was titrated and used for ChIP at dilution 1:500 and 1:100; the best results were obtained with the 1:100 dilution.

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-H4K20me3 crude serum (Product # 49-1024) with other histone modifications of histone H3 and histone H4. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di-, and trimethylation of adjacent lysines. To determine the cross reactivity, 0.2 to 100 pmol of peptide containing the respective histone modifications was spotted on a membrane. The crude serum was used at dilution 1:1,000. At this dilution, the limit of detection of the antibody is 5 pmol.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Dot Blot was used to check the specificity of the anti-H4K20me3 antibody (Product # 49-1024) with peptides containing other modifications of histone H3 and H4. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di- and trimethylation of lysines 9, 27 and 36 of H3. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:1,000. The figure shows a high specificity of the antibody for the modification of interest.