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Our Abpromise guarantee covers the use of ab5103 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration. PubMed: 20733033|
|PepArr||Use a concentration of 0.2 - 0.02 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|Flow Cyt||Use at an assay dependent concentration.
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
|ChIP/Chip||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
Abcam recommends using 3-5% milk as the blocking agent
|ChIP||Use at an assay dependent concentration.|
Loading Control: GAPDH
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab5103 overnight at 4°C. Antibody binding was detected using Goat anti Rabbit IR680 secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
ab5103 staining Histone H3 (citrulline 2 + 8 + 17) in Mouse bone marrow cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde and permeabilized in 0.1% Triton X-100 prior to blocking in 5% Goat serum for 2 hours at 25°C. The primary antibody was diluted 1/250 in PBS and incubated with the sample for 12 hours at 4°C. The secondary antibody was Alexa Fluor® 488-conjugated Goat anti-Rabbit polyclonal, diluted 1/500.
Nuclei were counterstained blue with DAPI.
All batches of ab5103 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - citrulline 2 + 8 + 17 peptide (ab32876), indicating that this antibody specifically recognises the Histone H3 - citrulline 2 + 8 + 17 modifications.
ab32876 - Histone H3 - citrulline 2 + 8 + 17
ab17566 - Histone H3 - unmodified
Rabbit polyclonal to Histone H3 (citrulline 2 + 8 + 17) used at 1/2000 dilution, after blocking with TBST 5% BSA. Purified histones run out with approximately 250 ng of each histone.
Lanes 1-3 contain Histone H3 (250 ng per lane)
Lane 1: PADI4 + Calcium
Lane 2: H3 + PADI4
Lane 3: H3 + PADI4 + Calcium
Lanes 4-5 contain bulk histones (250 ng per lane)
Lane 4: PADI4
Lane 5: PADI4 + Calcium
Lane 6: MCF7 cell extract
Lane 7: MCF7 cell extract (HA-PADI4)
Secondary antibody : anti-rabbit HRP from Sigma.
In WB ab5103 only recognizes human or bovine histone H3 when PADI4 and calcium are added.
ICC/IF image of ab5103 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab5103, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above .
Blots were developled with Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody