ab4076 (4µg/ml) staining Histone H3 in human placenta using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.
Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

All lanes : Anti-Histone H3 (phospho T32) antibody (ab4076) at 1 µg/ml

Lane 1 : Colcemid treated histone calf thymus lysate
Lane 2 : Untreated histone calf thymus lysate
Lane 3 : Colcemid treated histone calf thymus lysate with Human Histone H3 (phospho T32) peptide (ab14799) at 1 µg/ml
Lane 4 : Untreated calf thymus histone lysate with Human Histone H3 (phospho T32) peptide (ab14799) at 1 µg/ml
Lane 5 : Colcemid treated calf thymus histone lysate with Human Histone H3 (unmodified ) peptide (ab2623) at 1 µg/ml
Lane 6 : Untreated calf thymus histone lysate with Human Histone H3 (unmodified ) peptide (ab2623) at 1 µg/ml

Lysates/proteins at 20 µg per lane.


ab4076 recognises a band at 17 kDa, corresponding to Histone H3, in colcemid treated lysates indicating that it is a phospho-specific antibody. ab4076 is specifically blocked using the immunizing peptide (ab14799), but not the unmodified control peptide (ab2623).

This indicates that ab4079 is specifically recognising Phosphorylated T32 of Histone H3. ab4076 specifically recognises the phosphorylated form of Histone H3