All lanes : Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804) at 1/1000 dilution

Lane 1 : SET8 depleted lysate from U-2 OS (human bone osteosarcoma epithelial cell line), whole cell lysate
Lane 2 : G1-arrested lysate from U-2 OS whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 11 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

Depletion of Set8, a histone methyltransferase, delays S-phase progression and impairs DNA replication (PMID: 18166648). It also prevents the monomethylation of H4K20, which is normally mediated by Set8 (PMID: 18166648).

The lysates in lanes 1 & 2 were kindly provided by our collaborator Dr. Anja Groth, Biotech Research and Innovation Centre (BRIC). University of Copenhagen.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in human colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Histone H4 (unmodified K20) was immunoprecipitated from 0.35 mg of SET8 depleted U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate with ab227804 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227804 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: SET8 depleted U-2 OS whole cell lysate 10 μg (Input).

Lane 2: ab227804 IP in SET8 depleted U-2 OS whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227804 in SET8 depleted U-2 OS whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Depletion of Set8, a histone methyltransferase, delays S-phase progression and impairs DNA replication (PMID: 18166648). It also prevents the monomethylation of H4K20, which is normally mediated by Set8 (PMID: 18166648).

This lysate was kindly provided by our collaborator Dr. Anja Groth, Biotech Research and Innovation Centre (BRIC). University of Copenhagen.

All lanes : Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804) at 1/1000 dilution

Lane 1 : Unmodified recombinant H4 protein, 10 ng
Lane 2 : Recombinant mutant H4 chemically modified to mimic H4K20Me1, 10 ng
Lane 3 : Recombinant mutant H4 chemically modified to mimic H4K20Me2, 10 ng
Lane 4 : Recombinant mutant H4 chemically modified to mimic H4K20Me3, 10 ng
Lane 5 : Unmodified recombinant mutant H4 (K20 mutated to C), 10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 11 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

The recombinant proteins were His-tagged.

 

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in rat spleen is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in mouse spleen is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in human bladder cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab227804 (red), and 20 µl of protein A/G sepharose beads slurry (10 µl of sepharose A beads + 10 µl of sepharose G beads). 2 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach).