Anti-Histone H3 Citrulline R2+R8+R17 Antibody (ab5103)

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103)

Blocking buffer: 2% BSA

Gel type: MES

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103) at 1 µg/mL

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

Rat brain tissue lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

Exposure time: 1min

Western blot analysis of citrullinated histone H3 in mouse and rat brain tissue lysates.

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103)

Blocking buffer: 2% BSA

Gel type: MES

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103) at 1 µg/mL

Lane 1:

NIH/3T3 (Mouse embryo fibroblast cell line) nuclear lysate at 10 µg

Lane 2:

PC12 (Rat adrenal pheochromocytoma cell line) nuclear lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

Exposure time: 1min

Western blot analysis of citrullinated histone H3 in NIH/3T3 and PC12 cell lines.

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103)

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.

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103) at 0.2 µg/mL

Lane 1:

HL60 whole cell lysate (negative control) at 20 µg

Lane 2:

HL60 whole cell lysate + DMSO (solvent control) at 20 µg

Lane 3:

HL60 whole cell lysate + DMSO + Calcium Ionophore (positive control) at 20 µg

Secondary

All lanes:

Goat anti Rabbit IR680 at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa

Observed band size: 17 kDa

Western blot of Histone H3 citrullination (ab5103) in HL60 cells under different treatments.

Peptide Array - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103)

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

Ab5103 antibody strongly binds to Histone H3 citrullinated at positions 2, 8, and 17, but not to unmodified Histone H3.

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103)

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

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (ab5103) at 1 µg/mL

All lanes:

HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Ionophore treated Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa

Observed band size: 17 kDa

Exposure time: 30s

Western blot of Histone H3 (citrulline R2+R8+R17) in HL60 cells treated with DMSO and Calcium Ionophore