AP32611SU-N Methylated Lysine (Di- and Tri-) antibody

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Rabbit anti Human, Vertebrates Methylated Lysine (Di- and Tri-)


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  • AP32611SU-N

Product Description for Methylated Lysine (Di- and Tri-)

Rabbit anti Human, Vertebrates Methylated Lysine (Di- and Tri-).
Presentation: Serum
Product is tested for Dot blot, Western blot / Immunoblot.

Properties for Methylated Lysine (Di- and Tri-)

Product Category Primary Antibodies
Quantity 0.1 ml
Presentation Serum
Reactivity Hu, Ve
Applications Dot, WB
Clonality Polyclonal
Host Rabbit
Molecular weight 17 kDa
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Polyclonal Antibodies (de)

Datasheet Extract

KLH-conjugated, synthetic peptide containing the sequence (...AR[me2K]ST...) in which [me2K] corresponds to dimethyl-lysine 9 of human histone H3.
Application Immunoblot Analysis: A 1/5,000-1/10,000 dilution of this lot detected di/trimethylated Histone H3 in acid extracts from HeLa cells. No detection of unmodified recombinant histone H3 was observed (See Figure 1). 
Peptide Dot Blot analysis: This antibody has been reported by an independent laboratory to detect di/trimethylated lysines in histones. Serial dilutions of various methylated peptides were spotted onto PVDF and probed with this antibody (See Figure 2).
Background Post-translational modifications of proteins play critical roles in the regulation and function of many known biological processes. Proteins can be post-translationally modified in many different ways, and a common posttranscriptional modification of Lysine involves methylation. Lysine can be methylated once, twice or three times by lysine methyltransferases. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histone methyltransferases. Histones which are methylated on certain residues can act epigenetically to repress or activate gene expression.
The transcriptional repressor SUV39H1 can encode novel enzymes which selectively methylate histone H3 at lysine 9. SUV39H1 places a methyl marker on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state. Some studies have also speculated a stimulatory role for transcription by methylated histone lyside 4 due to its presence at active transcription sites.
Protocols Immunoblot Protocol
1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on an acid-extracted protein sample (see protocol below) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.
2. Block the blotted nitrocellulose in freshly prepared 3% nonfat dry milk in TBS (TBS-MLK) for 1 hour at room temperature with constant agitation.
3. Incubate the nitrocellulose with 1:5,000-1:10,000 dilution of anti-di/trimethyl-Lysine, pan, diluted in freshly prepared TBS-MLK for 2 hours with agitation at room temperature.
4. Wash the nitrocellulose twice with water.
5. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1/5000 dilution was used) in TBS-MLK for 30 minutes with agitation at room temperature.
6. Wash the nitrocellulose twice with water.
7. Wash the nitrocellulose in TBS-0.05% Tween®-20 for 10 minutes.
8. Rinse the nitrocellulose in 4-5 changes of water.
9. Use detection method of choice (enhanced chemiluminescence was used). 

Acid Extraction of Proteins from HeLa Cells
1. Grow cells to 70% confluency in DMEM supplemented with 10% FBS.
2. Scrape the cells from the plate.
3. Pellet the cells by centrifugation at 200 x g for 10 minutes.
4. Decant the supernatant fraction.
5. Suspend the cells with 10-15 volumes of PBS and centrifuge at 200 x g for 10 minutes.
6. Decant supernatant fraction (PBS wash).
7. Suspend the cell pellet in 5-10 volumes of lysis buffer.
8. Add hydrochloric acid to a final concentration of 0.2M (0.2N). Use polypropylene tubes.
9. Incubate on ice for 30 minutes.
10. Centrifuge at 11,000 x g for 10 minutes at 4°C.
11. Keep the supernatant fraction, which contains the acid soluble proteins, and discard the acid-insoluble pellet.
12. Dialyze the supernatant against 200 mL 0.1M (0.1N) acetic acid, twice for 1-2 hours each.
13. Dialyze three times against 200 mL H2O for 1 hour, 3 hours, and overnight, respectively. The protein can be quantified and lyophilized or stored at -70°C.
Lysis buffer:
10mM HEPES, pH 7.9
1.5mM MgCl2
10mM KCl
0.5mM DTT*
1.5mM PMSF*
*Add PMSF and DTT just prior to use of the buffer.
Storage Store undiluted (in aliquots) at -20°C.
Avoid repeated freezing and thawing.
Shelf life: one year from despatch.
0.05% Sodium Azide
Liquid Serum
Species Reactivity
Species reactivity (expected):
Species reactivity (tested):
Recognizes Di/Trimethylated Lysines.

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