- 49-1015 - Invitrogen Antibodies H3C2 antibody

Wurm S, Zhang J, Guinea-Viniegra J, García F, Muñoz J, Bakiri L, Ezhkova E, Wagner EF
Genes & development 2015 Jan 15;29(2):144-56

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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-H3K27me3S28p crude serum (Product # 49-1015), diluted 1:250 in TBS-Tween containing 5% milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the western analysis with the crude serum; lane 1 shows the same analysis after incubation of the crude serum with 750 pmol blocking peptide for 1 hour at room temperature.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: To determine the titer, an ELISA was performed using a serial dilution of anti-H3K27me3S28p crude serum (Product # 49-1015). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against dilution, the titer of the crude serum was estimated to be 1:8, 300.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Asynchronous HeLa cells were formaldehyde fixed, permeabilized with sodium citrate and Triton® X-100 (Dow Chemical Co) and blocked with PBS containing 2.5% BSA. (A) Cells were labeled with the anti-H3K27me3S28p crude serum (diluted 1:200 and incubated for 1 hour at room temperature) followed by labeled goat anti-rabbit secondary antibody (Fisher Scientific). (B) Nuclei were stained using the DNA-specific stain DAPI. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Asynchronous HeLa cells were formaldehyde fixed, permeabilized with sodium citrate and Triton® X-100 (Dow Chemical Co) and blocked with PBS containing 2.5% BSA. (A) Cells were labeled with the anti-H3K27me3S28p crude serum (diluted 1:200 and incubated for 1 hour at room temperature) followed by labeled goat anti-rabbit secondary antibody (Fisher Scientific). (B) Nuclei were stained using the DNA-specific stain DAPI. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP assays were performed using HeLa cells treated with colcemid, the anti-H3K27me3S28p crude serum (Product # 49-1015), and optimized PCR primer pairs for qPCR. Each ChIP assay used sheared chromatin from 10,000 cells and a volume of anti-H3K27me3S28p crude serum (1, 5, and 10 µl). Additionally, ChIP was performed after incubation of the crude serum with 5 nmol blocking peptide for 1 hour at room temperature. IgG (5 µg⁄IP) was used as negative IP control. qPCR was performed with primers for the promoters of the active genes c-fos and RPL30 and for the inactive gene MYOD. This figure shows the recovery, expressed as a percentage 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 the anti-H3K27me3S28p crude serum (Product # 49-1015) with peptides containing other modifications of histone H3 and H4 and with peptides containing unmodified sequences from histone H3. 100 to 0.2 pmol of the peptides was spotted on a membrane. The antibody was used at a dilution of 1:20,000. This figure shows a high specificity of the crude serum for the peptide containing the modifications of interest.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 µl of the anti-H3K27me3S28p crude serum. The immunoprecipitated proteins were analyzed by western blot with the crude serum diluted 1:500 in TBS-Tween containing 5% milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lanes 2 and 3, respectively.

Supportive validation

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-H3K27me3S28p crude serum (Product # 49-1015) with peptides containing other modifications of histone H3 and H4 and with peptides containing unmodified sequences from histone H3. 100 to 0.2 pmol of the peptides was spotted on a membrane. The antibody was used at a dilution of 1:20,000. This figure shows a high specificity of the crude serum for the peptide containing the modifications of interest.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 µl of the anti-H3K27me3S28p crude serum. The immunoprecipitated proteins were analyzed by western blot with the crude serum diluted 1:500 in TBS-Tween containing 5% milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lanes 2 and 3, respectively.

49-1015