Anti-Histone H1.2 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.2 antibody (AB17677)

ab17677 staining Histone H1.2 in HeLa cells. The cells were fixed with 100% methanol (5 min) at room temperature, permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tritonn for 1h. The cells were then incubated with the antibody ab16766 at 0.1μg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1hour at room temperature.

• IP

Unknown

Immunoprecipitation - Anti-Histone H1.2 antibody (AB17677)

Histone H1.2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5μg of Rabbit polyclonal to Histone H1.2 and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab17677.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 35kDa; Histone H1.2

All lanes:

Immunoprecipitation - Anti-Histone H1.2 antibody (ab17677)

Predicted band size: 21 kDa

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Exposure time: 20min

• WB

Lab

Western blot - Anti-Histone H1.2 antibody (AB17677)

False colour image of Western blot : Anti-Histone H1.2 antibody staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab17677 was shown to bind specifically to Histone H1.2. A band was observed at 32 kDa in wild-type HeLa cell lysates with no signal observed at this size in HIST1H1C knockout cell line ab261794 (knockout cell lysate ab257218). To generate this image, wild-type and HIST1H1C knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Histone H1.2 antibody (ab17677) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HIST1H1C (Histone H1.2) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-hist1h1c-histone-h12-knockout-hela-cell-lysate-ab257218'>ab257218</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 32 kDa,50 kDa

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• WB

Collaborator

Western blot - Anti-Histone H1.2 antibody (AB17677)

All lanes:

Western blot - Anti-Histone H1.2 antibody (ab17677) at 1/1000 dilution

Lane 1:

Recombinant histone H1

Lane 2:

Recombinant histone H1.1

Lane 3:

Recombinant histone H1.2

Lane 4:

Recombinant histone H1.3

Lane 5:

Recombinant histone H1.4

Lane 6:

Recombinant histone H1.5

Predicted band size: 21 kDa

Observed band size: 29 kDa

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This image is courtesy of Dr Albert Jordan and Monica Sancho, Center for Genomic Regulation (CRG). Recombinant H1 isoforms courtesy of Dr Nicole Happel.

• WB

Ap

Western blot - Anti-Histone H1.2 antibody (AB17677)

All lanes:

Western blot - Anti-Histone H1.2 antibody (ab17677) at 1 µg/mL

Lane 1:

Histone H1 Recombinant Protein at 0.1 µg

Lane 2:

Histone H1.2 (Human) - Recombinant Protein at 0.1 µg

Lane 3:

Histone H1 Recombinant Protein at 0.1 µg with Human Histone H1.2 peptide (<a href='/en-us/products/proteins-peptides/human-histone-h12-peptide-ab18500'>ab18500</a>)

Lane 4:

Histone H1.2 (Human) - Recombinant Protein at 0.1 µg with Human Histone H1.2 peptide (<a href='/en-us/products/proteins-peptides/human-histone-h12-peptide-ab18500'>ab18500</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 21 kDa

Observed band size: 29 kDa

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Exposure time: 12min

• WB

CiteAb

Western blot - Anti-Histone H1.2 antibody (AB17677)

Western Blotting using Anti-Histone H1.2 antibody, ab17677. Publication image from Li, Z. et al., 2018, Cell Res, 29844578. Legend direct from paper.

PARylation of H1.2 is essential for ATM activation. aParp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 µM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 µM etoposide for the indicated time with or without exposure to 5 µM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 µM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 µM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 µM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 µM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 µm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD+ for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD+ for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 µM etoposide for 1 h or 5 µM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

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• WB

CiteAb

Western blot - Anti-Histone H1.2 antibody (AB17677)

Western Blotting using Anti-Histone H1.2 antibody, ab17677. Publication image from Li, Z. et al., 2018, Cell Res, 29844578. Legend direct from paper.

Linker histone H1.2 inhibits ATM recruitment and activation by interacting with MRN. a Wild type and H1.2 KO (1#) HeLa cells were transfected with GFP-NBS1 and subjected to laser micro-irradiation-coupled live-cell imaging. Images were taken every 10 s for 10 min and the relative intensity of the irradiation path signal was shown. The data represent the mean ± SD. Scale bars, 10 µm. b HeLa cells extracts were analyzed by Co-IP assay with or without benzonase treatment with the indicated antibodies. c GST alone or GST-MRE11, RAD50 and NBS1 were incubated with HIS-H1.2 for GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-MRE11 for GST pull-down assay. * indicates specific protein bands. e Wild type or NBS1 KO HeLa cells were transfected with the indicated siRNAs and treated with 40 µM etoposide for 2 h and analyzed by immunoblotting. f HeLa cells were transfected with the indicated siRNAs and treated with 40 µM etoposide for 2 h and analyzed by immunoblotting. g, h HeLa cells were transfected with the indicated plasmids, and the whole cell lysates were immunoprecipitated with ATM antibody and analyzed by immunoblotting. i HeLa cells were transfected with the indicated plasmids and treated with 40 µM etoposide for 2 h. Whole cell extracts were prepared and analyzed by Co-IP assay and immunoblotting with the indicated antibodies

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• WB

CiteAb

Western blot - Anti-Histone H1.2 antibody (AB17677)

Western Blotting using Anti-Histone H1.2 antibody, ab17677. Publication image from Li, Z. et al., 2018, Cell Res, 29844578. Legend direct from paper.

PARylation of H1.2 is essential for ATM activation. aParp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 µM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 µM etoposide for the indicated time with or without exposure to 5 µM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 µM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 µM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 µM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 µM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 µm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD+ for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD+ for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 µM etoposide for 1 h or 5 µM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

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