Anti-Histone H2A.Z antibody - ChIP Grade (ab4174)

  Immunocytochemistry/ Immunofluorescence - Histone H2A.Z antibody - ChIP Grade (ab4174)

The staining has been done on mouse embryonic cells. The fixation is 2% PFA, and permeabilization is PBS 0.5% triton BSA. The dilution used was 1 in 100 (but it could be used at 1/200 to1/300)

Red  = H2A.Z
Blue  = toto3 for the DNA

  Western blot - Histone H2A.Z antibody - ChIP Grade (ab4174)

Lanes 1 - 2 : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1/500 dilution
Lanes 3 - 4 : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1/1000 dilution

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Histone H2A.Z peptide (ab11681) at 1 µg/ml
Lane 3 : Calf thymus histone lysate
Lane 4 : Calf thymus histone lysate with Histone H2A.Z peptide (ab11681) at 1 µg/ml

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 13.4 kDa
Observed band size : 15 kDa (why is the actual band size different from the predicted?)

  ChIP - Histone H2A.Z antibody - ChIP Grade (ab4174)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab4174 (blue), and 20 µl 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 and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H2A.Z antibody - ChIP Grade (ab4174)

IHC image of Histone H2A.Z staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4174, 5µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  Immunocytochemistry/ Immunofluorescence - Histone H2A.Z antibody - ChIP Grade (ab4174)

This image was kindly supplied as part of the review submitted by Dr Geza Fejes-Toth.

  Western blot - Histone H2A.Z antibody - ChIP Grade (ab4174)

All lanes : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 13.4 kDa
Observed band size : 16 kDa (why is the actual band size different from the predicted?)
Additional bands at : 36 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 3 minutes

  Immunocytochemistry/ Immunofluorescence - Histone H2A.Z antibody - ChIP Grade (ab4174)

ICC/IF image of ab4174 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4174, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.