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Synthetic peptide conjugated to KLH derived from within residues 100 - 200 of Human Histone H1.4, phosphorylated at T146.
(Peptide available as ab10139.)
Our Abpromise guarantee covers the use of ab3596 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000. Detects a band of approximately 32 kDa.Can be blocked with Histone H1 (phospho T146) peptide (ab10139).|
|ICC/IF||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
Rabbit polyclonal to Histone H1 phospho T146 (1/1000) against recombinant Histone H1, incubated with (control) untreated insect cell lysate (lanes 1, 3, 5, 7, 9, 11, 13) or insect cell lysate containing active cyclin E / CDK2 complexes (lanes 2, 4, 6, 8, 10, 12, 14).
Using site-directed mutagenesis mutant Histone H1 proteins were made. Five phosphorylation cyclin/cdk phosphorylation concensus sites were mutated : T18, T146, T154, S172 and S187.
Lanes 3-12 contain mutant histone H1 with only one wild-type cyclin/cdk phosphorylation concensus site (indicated in brakets).
Lanes 13-14 contain mutant Histone H1 with all 5 sites mutated to Ala.
Lanes 1-2 : wt Histone H1
Lanes 3-4 : T146A, T154A, S172A, S187A (wt site at T18)
Lanes 5-6 : T18A, T154A, S172A, S187A (wt site at T146)
Lanes 7-8 : T18A, T146A, S172A, S187A (wt site at T154)
Lanes 9-10 : T18A, T146A, T154A, S187A (wt site at S172)
Lanes 11-12: T18A, T146A, T154A, S172A (wt site at S187)
Lanes 13-14: T18A, T146A, T154A, S172A, S187A.
Lane two shows that Histone H1 is phosphorylated in the presence of cyclin E/ CDK2 complexes whilst in lane 1 the site is unphosphorylated and the ab doesn't recognise it.
Lane 6 contains a mutated H1 histone where all of the phosphorylatable residues are mutated with the exception of T146. This shows that the antibody recognises T146 residue. The two bands appear to be running at different levels but we believe that this is due to curvature of the gel.
Alejandro Contreras, Baylor College of Medicine
Rabbit polyclonal to Histone H1.4 (phospho T146) (1/1000).
Human neuroblastoma cell line SK-N-SH cultured on coverslips were fixed in 4% paraformaldehye and then stained with ab3596 (green). The DNA stained with DAPI is shown in red. (100x magnification).
ab3596 (2µg/ml) staining histone H1 Phospho T146 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of smooth muscle alongside nuclear and cytoplasmic staining of myenteric plexus.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
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