Western blot - CENPA antibody [3-19] - ChIP Grade (ab13939)

Predicted band size : 16 kDa

The antibody works well with histones from: Hela cells (human), Friend cells (mouse) and calf thymus (Roche). 5 µg acid-extracted histones were loaded per lane. Images taken following 5 min exposure. 1:1000 dilution.

Lane 1: Friend cells (mouse)

Lane 2: Hela cells (human)

Lane 3: Calf thymus

Jerome Govin (Grenoble)

Immunocytochemistry/ Immunofluorescence - CENPA antibody [3-19] - ChIP Grade (ab13939)

This picture shows human metaphase chromosomes detected with ab13939 as primary antibody and AF488 goat anti-mouse secondary antibody (green). This image was kindly supplied as part of the review submitted by Professor Beth A. Sullivan, Boston University School of Medicine.

ChIP - CENPA antibody [3-19] - ChIP Grade (ab13939)

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 25µg of chromatin, 4µg of  ab13939 (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 and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

Immunocytochemistry/ Immunofluorescence - CENPA antibody [3-19] - ChIP Grade (ab13939)

ab13939 staining CENPA by Immunofluorescence. This antibody works very well in HeLa cells, with double staining of kinetochores using CREST indicating perfect co-localization.

Optimal antibody dilution: 2mg/ml.

Optimal fixation protocol: PFA fixation: 10 min in 3,7 % PFA  at room temperature, 5 min in PBS / 0,1%Triton X-100,  then 3 washes in PBS.

Also works well after PFA/Methanol fixation: 10 min in 3,7% PFA Troom, 1 wash in PBS, MetOH 20 min, -20°C, then 3 washes in PBS.

IF staining was performed following a standard protocol:  blocking: 30 min; primary antibody: 1 hr; secondary antibody: 45 min. All incubations were at 37 °C in PBS/ 0,1% Tween containing 3% BSA.

Image and experimental conditions kindly provided by Serena Orlando, Giulia Guarguaglini and Patrizia Lavia, of the University 'La Sapienza' CNR, Italy

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-CENPA antibody [3-19] - ChIP Grade(ab13939)

IHC image of ab13939 staining in normal human skin formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab13939, 5µg/ml, for 15 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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Flow Cytometry-CENPA antibody [3-19] - ChIP Grade(ab13939)

Overlay histogram showing HeLA cells stained with ab13939 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab13939, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was Mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.