Anti-Histone H1 (phospho T146) antibody

• Supplier: Abcam
• Catalog: ab3596
• Prices: $401.00
• Sizes: 50 µg
• Host: Rabbit
• Clonality: Polyclonal
• Isotype: IgG
• Applications: WB ICC/IF ICC/IF IHC-P
• Species Reactivities: Human, Mouse
• Antigen: Synthetic peptide conjugated to KLH derived from within residues 100 - 200 of Human Histone H1
• Description: Rabbit Polyclonal
• Gene: HIST1H1E
• Conjugate: Unconjugated

Product images

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 H1Lanes 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, S1

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).

All lanes : Anti-Histone H1 (phospho T146) antibody (ab3596) at 1 µg/mlLane 1 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treatedLane 2 : HeLa Histone Preparation Nuclear LysateLane 3 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated (ab30751) with Histone H1 (phospho T146) peptide (ab10139) at 1 µg/mlLane 4 : HeLa Histone Preparation Nuclear Lysate with Histone H1 (phospho T146) peptide (ab10139) at 1 µg/mlLane 5 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated (ab30751) with Human Histone H1 peptide (ab30741) at 1 µg/mlLane 6 : HeLa Histone Preparation Nuclear Lysate with Human Histone H1 peptide (ab30741) at 1 µg/mlLysates/proteins at 2.5 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.Observed band size : 32 kDa (why is the actual band size different from the predicted?)Additional bands at : 110 kDa. We are unsure as to the identity of

ICC/IF image of ab3596 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3596, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa, Hek293 and HepG2 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.

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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