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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- ELISA [1]
- Immunocytochemistry [1]
- Protein array [1]
- Chromatin Immunoprecipitation [2]
- Other assay [1]
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- Product number
- PA5-40103 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- H2BK15ac Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 50 µg
- Concentration
- 2.1 mg/mL
- Storage
- -20°C or -80°C if preferred
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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-H2BK15ac antibody (Product # PA5-40103). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.
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-H2BK15ac antibody (Product # PA5-40103) 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 1:29,700.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- HeLa cells were stained with the anti-H2BK15ac antibody (Product # PA5-40103) 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 H2BK15ac 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.
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 H2B (Lys15) polyclonal antibody (Product # PA5-40103) with peptides containing other histone modifications and the unmodified H2B. 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.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 2 µg of Acetyl-Histone H2B (Lys15) polyclonal antibody (Product # PA5-40103). The IP'd DNA was subsequently analyzed on a HiSeq. 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- ChIP assays were performed on human HeLa cells using a Acetyl-Histone H2B (Lys15) polyclonal antibody (Product # PA5-40103) 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).
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 H2B (Lys15) polyclonal antibody (Product # PA5-40103) with peptides containing other histone modifications and the unmodified H2B. 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.
