Anti histone h4 mono methyl r3 antibody

Nuclear acid proteins were separated on 18 % polyacrylamide SDS-PAGE gel while nuclear extract were separated on 10 % polyacrylamide SDS-PAGE gel and transferred to a nitrocellulose membrane according to the protocol described by Towbin et al., 1979 [12 (link)]. The membrane was exposed to Ponceau S to verify the efficiency of the transfer. The membrane was blocked with 5 % milk in PBS buffer- Tween-20 0.05 % (PBS-T) for 2 h at room temperature and then incubated with the anti-histone H3 antibody (Abcam ab1791) 1:5000, anti-histone H4 antibody (Santa Cruz sc-8658-R) 1:2000, anti-acetyl-histone H4 (Millipore 06-866) 1:4000, anti-histone H4 acetyl K12 antibody (Abcam ab61238) 1:2000, anti-histone H4 mono methyl R3 antibody (Abcam ab17339) 1:500, anti-trimethyl-histone H4 (Lys20) antibody (Millipore 07-463) 1:500, anti-lamin B1 antibody (Abcam ab16048) 1:5,000, anti-fibrillarin 1:3,000, anti-PRP6 1:500, all diluted with 2 % milk PBS-T overnight at 4 °C. The membranes were rinsed 3 times with PBS-T and incubated for 2 h with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific G-21234) 1:7,500 or goat anti-mouse IgG antibody (Thermo Fisher Scientific G-21040) 1:7,500 diluted in 2 % milk PBS-T. The antibody staining reaction on the membranes were developed by SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific 34095).

Trophozoites in a logarithmic growth phase were harvested and transferred on glass coverslips coating with poly L - lysine and incubated for 3 h at 37 °C to let them attach to the glass surface. An indirect immunofluorescence assay was performed as follows. Amoebas were fixed and permeabilized with cold methanol-acetone 50:50 for 10 min at room temperature and washed twice with PBS buffer. After, trophozoites were incubated for 1 h with 1 % bovine serum albumin in PBS buffer and samples were reacted with anti-acetyl-histone H4 (Millipore 06-866) 1:1500, anti-histone H4 acetyl K12 antibody (Abcam ab61238) 1:300, anti-histone H4 mono methyl R3 antibody (Abcam ab17339) 1:200, anti-5-methylcytosine antibody (Abcam ab10805) 1:25, anti-lamin B1 antibody (Abcam ab16048) 1:200, anti C23 (H-250) antibody (Santa Cruz sc-13057) 1:100, anti-fibrillarin 1:50 and anti-PRP6 1:50 overnight at 4 °C. Then washed with PBS and incubated for 1 h at 37 °C with Alexa Fluor® 568 goat anti-rabbit IgG (Invitrogen A11036) 1:200 and Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen A-11001) 1:100. Nuclei were stained with 4,6-Diamidino-2-Phenylindole (DAPI) Vectashield Mounting (Vector H-1200) and samples were observed through a confocal microscope Fluoview Olympus FV300. Software 4.3.