Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab10158 (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.

All lanes : Anti-Histone H4 antibody - ChIP Grade (ab10158) at 1 µg/ml

Lane 1 : Histone prep
Lane 2 : HeLa histone lysate
Lane 3 : Histone prep with Human Histone H4 peptide (ab13843) at 1 µg/ml
Lane 4 : HeLa Histone lysate with Human Histone H4 peptide (ab13843) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 11 kDa

IHC image of Histone H4 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 ab10158, 1µg/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.

Histone H4 was immunoprecipitated using 0.5mg NIH/3T3 whole cell lysate, 5µg of Rabbit polyclonal to Histone H4 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, NIH3T3 whole cell lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab10158.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 14kDa: Histone H4; non specific - 52 and 85kDa: We are unsure as to the identity of this extra band.

All lanes : Anti-Histone H4 antibody - ChIP Grade (ab10158) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 11 kDa
Observed band size: 14 kDa

why is the actual band size different from the predicted?