Western blot - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] (ab6000)

All lanes : Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) at 1/500 dilution

Lane 1 : Histone Prep
Lane 2 : Histone Prep with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml
Lane 3 : Histone Prep with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
Lane 4 : Histone Prep with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
Lane 5 : Histone Prep with Histone H3 peptide - mono methyl K27 (ab1780) at 1 µg/ml
Lane 6 : Histone Prep with Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
Lane 7 : Histone Prep with Histone H3 peptide - tri methyl K9 (ab1773) at 1 µg/ml
Lane 8 : Histone Prep with Histone H3 peptide - unmodified (ab2903) at 1 µg/ml

Lysates/proteins at 1 µg per lane.

Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 15.2 kDa


Exposure time : 2 minutes

Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

Immunofluorescent imaging of human cells (U2OS) with ab6000 reveals a diffuse background nuclear staining corresponding to global dimethylation of K4, with multiple foci of brighter staining.  The complete lack of nucleolar or cytoplasmic staining background confirms the high specificity of this antibody in this application.   

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees.

Due to the size constraints for images on our website, unfortunately we are unable to show a higher quality image.

Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

ChIP - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

Chromatin was prepared from U2OS 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  ab6000 (blue), and 20 µl 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). Primers and probes are located in the first kb of the transcribed region.

Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

ICC/IF image of ab6000 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6000, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 and MCF7 cells at 1µg/ml.

Immunofluorescence - Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

Left image shows human Hep cell monolayer fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100, blocked with 5% FCS in PBS (20 min) and incubated with ab6000 for 1 hr (diluted 1/200 in PBS plus 1% FCS). Detection with anti-mouse rhodamin labeled secondary antibody. Unstained areas represent nucleoli and heterochromatin as judged from DAPI overlay of the same cell in the right image.

This image was kindly supplied as part of the review submitted by Christian Schoefer.