Anti-Histone H4 (symmetric di methyl R3) 抗体

• WB

Lab

Western blot - Anti-Histone H4 (symmetric di methyl R3) antibody (AB5823)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab5823 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H4 (symmetric di methyl R3) antibody (ab5823) at 1 µg

All lanes:

Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (<a href='/products/secondary-antibodies/vhh-single-domain-rabbit-igg-fc-hrp-ab191866'>ab191866</a>) at 1/50000 dilution

Predicted band size: 11 kDa

Observed band size: 14 kDa

true

Exposure time: 3min

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (symmetric di methyl R3) antibody (AB5823)

IHC image of ab5823 staining Histone H4 (symmetric di methyl R3) in human kidney formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab5823, 5μg/ml, for 15 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. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• PepArr

Lab

Peptide Array - Anti-Histone H4 (symmetric di methyl R3) antibody (AB5823)

All batches of ab5823 are tested in Peptide Array against peptides to different Histone H4 and H2A modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H4 - symmetric di methyl R3 peptide (ab14791), indicating that this antibody specifically recognises the Histone H4 - symmetric di methyl R3 modification.

ab14791 - Histone H4 - symmetric di methyl R3

ab17416 - Histone H4 asymmetric di methyl R3

ab15821 - Histone H4 - unmodified

ab22399 - Histone H2A symmetric di methyl R3

ab22398 - Histone H2A asymmetric di methyl R3

ab13186 - Histone H2A - unmodified

• WB

CiteAb

Western blot - Anti-Histone H4 (symmetric di methyl R3) antibody (AB5823)

Western Blotting using Anti-Histone H4 (symmetric di methyl R3) antibody, ab5823. Publication image from Liu, M. et al., 2020, Theranostics, 32292506. Legend direct from paper.

PRMT5-dependent direct interaction with c-Myc represses gene expression of PTEN and p57. (A) The subcellular location of c-Myc and PRMT5 proteins was documented in BGC823 and SGC7901 cells by immunofluorescence microscopy. Scale bar : 10 µm. (B) Co-immunoprecipitation of endogenous c-Myc with Flag-PRMT5 from SGC7901 cells overexpressing Flag-tagged PRMT5. IgG was used as the negative control. (C) Western blot analysis of c-Myc binding to purified GST or GST-PRMT5 fusion protein using c-Myc antibody (top). GST or GST-PRMT5 fusion protein purified from E. coli was visualized by Coomassie blue staining (bottom). An asterisk denotes the GST-PRMT5 fusion protein. (D) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5 fragments F1 (amino acids 1-354), F2 (amino acids 355-453) or F3 (amino acids 454-637) using c-Myc antibody (top). GST or GST-PRMT5 F1, F2, or F3 fusion proteins from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST-PRMT5 F1, F2, and F3 fusion proteins. (E) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5 or GST-PRMT5 488-494δ (deletion of amino acids 488-494) using c-Myc antibody (top). GST, GST-PRMT5 or GST-PRMT5 488-494δ purified from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST-PRMT5 or GST-PRMT5 488-494δ fusion proteins. (F) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5, GST-PRMT5 R488A or GST-PRMT5 K490A using c-Myc antibody (top). GST, GST-PRMT5, GST-PRMT5 R488A or GST-PRMT5 K490A purified from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST- PRMT, GST-PRMT5 R488A or GST-PRMT5 K490A fusion proteins. (G) Western blot analysis of H4R3me2s levels in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. Histone H4 served as a loading control. (H) Relative mRNA levels of PRMT5, PTEN, p57, p18, p21 and p63 was examined by quantitative real-time PCR assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. GAPDH was used as an endogenous control. *P < 0.05, **P < 0.01. (I) Protein levels of PRMT5, PTEN, p57, p18, p21 and p63 were examined by Western blot assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. GAPDH served as a loading control. (J) Relative enrichment of H4R3me2s at the promoters of PTEN (P4, left panel) and p57 (P5, right panel) was examined by ChIP assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. IgG was used as a negative control. Data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01. (K) Relative enrichment of H4R3me2s at the promoters of PTEN (P4, left panel) and p57 (P5, right panel) was examined by ChIP assays in NC, c-Myc siR-1 and c-Myc siR-2-treated BGC823 cells. IgG was used as a negative control. Data shown are mean ± SD (n = 3). **P < 0.01.

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