Histone H3, phosphorylated, trimethyl (Thr3/Lys4, Thr22/Lys23)

Applications: \nSuitable for use in Peptide Dot Blot, Peptide inhibition, Western Blot and Immunoprecipitation. Other applications not tested.\n\nRecommended Dilution:\nPeptide Dot Blot Analysis: 0.2ug/ml detects phospho-trimethyl (Thr3/Lys4 & Thr22/Lys23) histone H3. Slight crossreactivity was seen with phospho-dimethyl Thr3/Lys4 histone H3.\n\nPeptide Inhibition: Specificity was confirmed by the ability of 1uM or 5uM of the immunizing peptide to completely abolish detection of histone H3 in immunoblot analysis of HeLa acid extracts. HeLa acid extracts were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-phosphotrimethyl (Thr3/Lys4 & Thr22/Lys23) Histone H3 (0.2ug/ml) or anti-phospho-trimethyl (Thr3/Lys4 & Thr22/Lys23) Histone H3 pre-absorbed with 1uM and 5uM histone H3 peptides.\n\nWestern Blot: 0.2ug/ml detects phospho-trimethyl Histone H3 (Thr3/Lys4 & Thr22/Lys23) in acid extracted proteins from colcemid treated HeLa cells. No signal was detected in unmodified recombinant histone H3. Acid extracts from colcemid treated and untreated HeLa cells were resolved by electrophoresis, transferred to nitrocellulose and probed with antiphospho-trimethyl (Thr3/Lys4 & Thr22/Lys23) Histone H3. Proteins were visualized using a goat antirabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.\n\nImmunoprecipitation: 4ug immunoprecipitated phosphorylated and trimethylated histone H3 from 250ug of acid extracted proteins from HeLa cells.\n\nOptimal dilutions to be determined by the researcher.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

H5110-11Y

Size

200ug

Applications

IP, WB

Hosts

Rabbit

Reactivities

Hum, Mouse

Form

Supplied as a liquid in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide before the addition of glycerol to 30%.

P Type

Pab

Purity

Purified by Protein A affinity chromatography.

Isotype

IgG

Additional Info

Recognizes dual-modified phosphotrimethyl histone H3 (Thr3/Lys4 & Thr22/Lys23), Mr ~17kD. Slight crossreactivity with phospho(Thr3) dimethyl(Lys4) histone H3, trimethyl(Lys79) phospho(Thr80) histone H3, and trimethyl(Lys4) histone H3. Species Crossreactivity: Human and mouse. Broad species crossreactivity expected.