PA5-40102

antibody from Invitrogen Antibodies

Targeting: H2AC11 H2A.1b, H2A/p, H2AFP, HIST1H2AG, pH2A/f

Western blot (Supportive data in Antibodypedia)

ELISA (Supportive data in Antibodypedia)

Protein array (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-40102

Provider

Invitrogen Antibodies

Product name

H2AK5ac Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

50 µg

Concentration

0.93 mg/mL

Storage

-20°C or -80°C if preferred

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the anti-H2AK5ac antibody (Product # PA5-40102). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. 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 of the antibody, an ELISA was performed using a serial dilution of the anti-H2AK5ac antibody (Product # PA5-40102) in antigen coated wells. 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 1:25,000.

Protein array

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

A Dot Blot analysis was performed to test the cross reactivity of Acetyl-Histone H2A (Lys5) polyclonal antibody (Product # PA5-40102) with peptides containing other histone modifications and the unmodified H2AK5. 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 3 shows a high specificity of the antibody for the modification of interest.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 0.5 µg of Acetyl-Histone H2A (Lys5) polyclonal antibody (Product # PA5-40102). The IP'd DNA was subsequently analyzed on a HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment along the complete sequence and a 1 Mb region of the X-chromosome (fig 2A and B) and in genomic regions of chromosome 7, surrounding the ACTB gene, and of chromosome 12, surrounding the GAPDH gene (fig 2C and D). The position of the amplicon used for ChIP-qPCR is indicated by an arrow.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP assays were performed on human HeLa cells using a Acetyl-Histone H2A (Lys5) polyclonal antibody (Product # PA5-40102) and optimized PCR primer sets for qPCR. ChIP was performed with sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 0.5, 1, 2 and, 5 µg per ChIP experiment was analyzed. IgG (1 µg/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the GAPDH and ACTB promoters, used as positive controls, and for the coding region of the inactive MYOD1 gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % 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 Acetyl-Histone H2A (Lys5) polyclonal antibody (Product # PA5-40102) with peptides containing other histone modifications and the unmodified H2AK5. 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 3 shows a high specificity of the antibody for the modification of interest.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

A Dot Blot analysis was performed to test the cross reactivity of Acetyl-Histone H2A (Lys5) polyclonal antibody (Product # PA5-40102) with peptides containing other histone modifications and the unmodified H2AK5. 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 3 shows a high specificity of the antibody for the modification of interest.