重组Anti-Histone H4 (tri methyl K20)抗体[EPR17001(2)] - ChIP Grade (ab177190)

Dot blot analysis of Histone H4 (tri methyl K20) labeled with ab177190 at 1/1000 dilution.
Lane 1: Human H4 peptide a
Lane 2: Human H4 peptide c
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as secondary antobody at 1/100000 dilution.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 180 minutes.

ab177190 (0.005μg/mL) was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). A Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1/50000 was used as the secondary antibody. Protein Array Washing Buffer, 2*10 mins and 2*5 mins. Blocking buffer was 5% BSA in TBST and Innoscan 710 scanner was used. Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. 

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab177190 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active loci and Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

5% NFDM/TBST: Blocking and diluting buffer.

Dot blot analysis of Histone H4 (tri methyl K20) peptide(aa16-25) (Lane 1) and unmodified Histone H4 peptide (aa 16-25) (Lane 2) labeled using ab177190 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H4 (tri methyl K20) using ab177190 at 1/8000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Nucleus staining on Human colon was observed. Counterstained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H4 (tri methyl K20) using ab177190 at 1/8000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Nucleus staining on mouse colon was observed. Counterstained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H4 (tri methyl K20) using ab177190 at 1/8000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Nucleus staining on rat colon was observed. Counterstained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.