All lanes : Anti-Histone H2A.Z antibody [EPR6171(2)(B)] - ChIP Grade (ab150402) at 1/5000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 3 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysates
Lane 4 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 5 : C6 (Rat glial tumor glial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 13 kDa
Observed band size: 13 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast tissue sections labeling Histone H2A.Z with purified ab150402 at 1/2000 dilution (1.09 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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 25 µg of chromatin, 2 µg of ab150402 (red), and 20 µl of Protein A/G sepharose beads. 2 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Histone H2A.Z with purified ab150402 at 1/200 dilution (10 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H2A.Z (red) with purified ab150402 at a 1/2500 dilution. Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat cerebrum tissue sections labeling Histone H2A.Z with purified ab150402 at 1:2000 dilution (1.09 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse cerebrum tissue sections labeling Histone H2A.Z with purified ab150402 at 1/2000 dilution (1.09 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 ab150402 (unpurified) (blue), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow).  The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H2A.Z with ab150402 (unpurified) at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunofluorescent analysis of HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Histone H2A.Z with ab150402 (unpurified) at 1/100 dilution.

All lanes : Anti-Histone H2A.Z antibody [EPR6171(2)(B)] - ChIP Grade (ab150402) at 1/1000 dilution (unpurified)

Lane 1 : Recombinant Human octamers containing H2A at 1 µg
Lane 2 : Recombinant Human octamers containing H2A at 0.5 µg
Lane 3 : Native recombinant octamers K562 cells at 3 µg
Lane 4 : Native recombinant octamers K562 cells at 1.5 µg
Lane 5 : Recombinant Human octamers containing H2A.Z.2.1 at 0.5 µg
Lane 6 : Recombinant Human octamers containing H2A.Z.1 at 0.5 µg

Secondary
All lanes : HRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 13 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 5 minutes

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Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling Histone H2A.Z with ab150402 (unpurified) at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human cervical carcinoma tissue using ab150402 (unpurified) showing positive staining.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded normal human kidney tissue using ab150402 (unpurified) showing positive staining.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human prostate hyperplasia tissue using ab150402 showing positive staining.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human ovarian carcinoma tissue using ab150402 (unpurified) showing positive staining.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.