Chromatin was prepared from HEK-293T transfected with Histone H3.3(mutated G34 R) and HEK-293T transfacted with Histone H3.3(WT) cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab254402 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human giant cell tumor of bone (PMID: 29241742). The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

All lanes : Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containi a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag®, whole cell lysate
Lane 4 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containngi a myc-His-tag®, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: 26 seconds. 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (Human embryonic kidney epithelial cell) (transfected with myc-tagged Histone H3.3 WT expression vector) (Left) or myc-tagged Histone H3.3 G34R expression vector (Right) cells labelling Histone H3.3 (mutated G34 R) with ab254402 at 1/5000 dilution (0.01ug) (Both panels). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Histone H3.3 (mutated G34 R) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) (transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®) whole cell lysate with ab254402 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254402 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate 10 ug

Lane 2: ab254402 IP in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254402 in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 41 seconds.

 

Dot blot analysis of Histone H3.3 (mutated G34 R) labeled with ab254402 at 1/1000 dilution.

Lane 1: Histone H3.3 H3G34R peptide (aa28-40).
Lane 2: Histone H3.3 H3G34R peptide (aa26-36).
Lane 3: Histone H3.3 WT peptide (aa26-40).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human testis. The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.