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Histones are nuclear proteins that form octameric structures which bind DNA to form units of chromatin called nucleosomes. The family of histones¡ªH2A, H2B, H3, and H4¡ªare key players in gene regulation. They undergo a number of post-translational modifications (PTM) in response to various stimuli, including phosphorylation on serine and threonine residues and methylation on lysine residues. PTMs produce configural changes in histone proteins that may induce nucleosome remodeling and expose or hide DNA sequences from transcriptional complexes. Histone H4 lysine 20 (H4K20) may undergo mono-, di-, or trimethylation, which is catalyzed by the methyltransferase PR-Set7 (Set8 or KMT5a). Methylated H4K20 plays a role in regulating DNA damage responses, mitosis, DNA replication, and gene expression. Trimethylation of H4K20 contributes to gene silencing, and is a mark of the repressive heterochromatin state.
1. HOW DID OUR ANTIBODY PERFORM? CUSTOMER REVIEW: Histone H4 (Tri Methyl Lys20) Polyclonal Antibody (STJ97154)
2. WHAT WAS USED? Primary antibody: STJ97154. Histone H4 (Tri Methyl Lys20) Polyclonal Antibody. Provider: St John’s Laboratory Dilution ratio: 1:500 Application: Western blot Materials for Validation: Drosophila Schneider-2 cell line Treatment of Materials: When cells were cultured to 70%-80% plating density, lysisbuffer with protease inhibitors (Sigma) and equal volume of 2X loading buffer were added for 10 min water bath at 85¡ãC. 16 kDa 6 kDa 4 kDa
3. WHAT WAS THE PROTOCOL? Gel electrophoresis: 4-15% polyacrylamide gel, constant voltage 150 V, 60 min. Transfer: 0.2 um nitrocellulose membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min. Blocking: 1X Odyssey buffer with PBS + 0.5% skimmed milk powder for 30 minutes. Primary antibody probing: primary antibody was added to the solution of blocking buffer, 4¡ãC for 2 nights. Membrane wash: 1X PBS wash for 3 times. Secondary antibody probing: secondary antibody (Li-cor donkey anti- rabbit) was added to the blocking buffer with 1:10000 dilution ratio, for 1 hr. Membrane wash: 1X PBS wash for 3 times. Visualization: Odyssey scanner. 16 kDa 6 kDa 4 kDa
4. WHAT WAS THE PROTOCOL? Gel electrophoresis information£º4-15% polyacrylamide gel, constant voltage 150 V, 60 min. Transfer information£º0.2 um nitrocellulose membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min. No. Antigen Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target Band Visualisation Time 1 Schneider-2 nuclear lysate STJ97154. Histone H4 (TriMethyl Lys20) Polyclonal Antibody 1:500 1:10 000 ~14 kDa N/A 16 kDa 6 kDa 4 kDa
5. WHAT DID THE CUSTOMER THINK? Testing results were provided by Serafin Colmenares from Lawrence Berkeley National Laboratory. They stated that ¡°Western blot produced band of expected size in Schneider-2 nuclear lysate.¡±
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