ChIP - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 5µg of  ab6002 (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.
    

Western blot - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

All lanes : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide (ab17163) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K4 (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K9 (ab1772) at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K27 (ab1781) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Exposure time : 3 minutes

Western blot - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

All lanes : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with BSA BLOCK
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with MILK BLOCK
Lane 3 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with BSA BLOCK
Lane 4 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with BSA BLOCK
Lane 5 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with MILK BLOCK
Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with MILK BLOCK

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Exposure time : 12 minutes

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Immunofluorescent imaging of human cells (U2OS) with ab6002 reveals broadly dispersed interphase nuclear staining corresponding to trimethylation of K27, with multiple foci of brighter staining, exactly agreeing with published studies of K27-trimethyl IF [See figure 3 of Peters et al Mol Cell 12(6):1577-1589 (2003)] .  The lack of nucleolar or cytoplasmic staining background confirms the high specificity of the 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 C. 

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

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Figure showing the nuclear distribution of H3 (tri-methyl K27) antibody, ab6002 in a) a 46 chromosome, XX cell line, and b) a 49 chromosome, XXXXX cell line.

The location of facultative heterochromatin at the inactive X chromosome is indicated by white arrow heads.

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Interphase 10T1/2 mouse fibroblasts were paraformaldehyde fixed (4%), immunofluorescently labeled with anti-trimethyl K27 antibody (ab6002) and counterstained with DAPI.  The merge image presents the DAPI and ab6002 channels as red and green, respectively.  The scale bar represents 3µm.

Kirk Mcmanus

ELISA - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

ELISA using ab6002 at antibody concentrations between 1/500 and 1/8000.

The red line indicates binding to the tri methyl K27 peptide (ab1782). Binding to the following peptides was not seen:
Unmodified K27 (ab2623),
mono methyl K27 (ab1780),
di methyl K27 (ab1781),
mono methyl K9 (ab1771),
di methyl K9 (ab1772),
tri methyl K9 (ab1773),
mono methyl K4 (ab1340),
di methyl K4 (ab7768),
tri methyl K4 (ab1342).

This indicates the specificity of ab6002 for tri methyl K27 of Histone H3.

Flow Cytometry - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

ab6002 staining mouse embyronic stem cells by flow cytometry. The ES cell colonies were trypsinized and permeabilized prior to blocking and staining with the antibody (1ug/1.5 x 10 ⁵ cells. A Cy3 conjugated anti-mouse antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Prof Albrecht Müller

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

ICC/IF image of ab6002 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 (ab6002, 5µg/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) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.

ChIP - Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Chromatin was prepared from nuclear lysate of the mouse embryonic stem cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in 1% formaldehyde. The primary antibody was diluted to 0.0133µg/µg chromatin and incubated in ChIP Sonication Buffer with the sample for 24 hours at 4°C. The immunoprecipitated DNA was quantified by real time PCR.

Cdx2: PCR primers situated in the promoter regions of Caudal type homeobox transcription factor 2
Nef3: PCR primers situated in the promoter regions of neurofilament 3

This image is a courtesy of Steve Bilodeau

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

ICC/IF image of ab6002 stained HepG2 cells. The cells were 100% methanol fixed (5 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 (ab6002, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 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.