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Tri-Methyl-Histone H4 (Lys20) Rabbit mAb

Catalog No.: F1267

Lane 1: Hela
Lane 2: NIH3T3

3 Customer Reviews

Experiment Essentials

Subcellular Location: Chromosome, Nucleosome核心, Nucleus.
WB
Recommending using RIPA/Nuclear Lysis Buffer to prepare lysates.
Recommended SDS-PAGE separating gel concentration: 20%.
Recommended wet transfer conditions: 200 mA, 60 min，Recommended to use 0.22 μm PVDF membrane.

Usage Information

Dilution
WB1:1000 IP1:50-1:200 CHIP1:50

Application
WB, ChIP

Source
Rabbit

Reactivity
Human, Mouse, Rat, Monkey, Xenopus, Bovine, Pig

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Storage (from the date of receipt)
–20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
11 Kda

Positive Control	Hela; NIH3T3
Negative Control

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 20%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 575. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 20%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

575. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
Tri-Methyl-Histone H4 (Lys20) Rabbit mAb recognizes endogenous levels of histone H4 protein only when tri-methylated at Lys20. This antibody does not cross-react with non-methylated, mono-methylated or di-methylated histone H4 Lys20. This antibody detects a 95 kDa non-specific protein of unkown origin.

Synonym(s)
Histone H4 (tri methyl K20),Tri-Methyl-Histone H4 (Lys20)

Uniprot ID
P62805

Clone
J13H2

Background
Tri-Methyl-Histone H4 (Lys20), also known as H4K20me3, is a specific histone post-translational modification crucial for maintaining genomic integrity and regulating chromatin structure. Histone H4 lysine 20 methylation exists in three states: mono-methylation (H4K20me1), di-methylation (H4K20me2), and tri-methylation (H4K20me3), with H4K20me3 being the trimethylation mark on the lysine 20 residue of histone H4. This modification is evolutionarily conserved from yeast to humans and serves as a marker for transcriptionally silent heterochromatin, indicating regions of the genome that are not actively transcribed. H4K20me3 is highly enriched at specific genomic locations such as pericentric heterochromatin, telomeres, imprinted regions, and repetitive elements, playing a role in maintaining these areas in a repressed state. It is essential for genomic integrity both in the absence and presence of genotoxic stress, forming part of the broader DNA damage response (DDR) network. The enzymes SUV4-20H1 and SUV4-20H2 primarily mediate the tri-methylation of H4K20. The different methylation states of H4K20 change dynamically throughout the cell cycle. H4K20me3 remains more stable and shows less dramatic changes compared to H4K20me1. H4K20me3 enhances chromatin folding and plays a structural role in the chromatin framework. Proper regulation of H4K20 methylation is vital for maintaining chromatin structure and function, as disruptions in this regulation result in genomic instability and other cellular dysfunctions.

Background

Tri-Methyl-Histone H4 (Lys20), also known as H4K20me3, is a specific histone post-translational modification crucial for maintaining genomic integrity and regulating chromatin structure. Histone H4 lysine 20 methylation exists in three states: mono-methylation (H4K20me1), di-methylation (H4K20me2), and tri-methylation (H4K20me3), with H4K20me3 being the trimethylation mark on the lysine 20 residue of histone H4. This modification is evolutionarily conserved from yeast to humans and serves as a marker for transcriptionally silent heterochromatin, indicating regions of the genome that are not actively transcribed. H4K20me3 is highly enriched at specific genomic locations such as pericentric heterochromatin, telomeres, imprinted regions, and repetitive elements, playing a role in maintaining these areas in a repressed state. It is essential for genomic integrity both in the absence and presence of genotoxic stress, forming part of the broader DNA damage response (DDR) network. The enzymes SUV4-20H1 and SUV4-20H2 primarily mediate the tri-methylation of H4K20. The different methylation states of H4K20 change dynamically throughout the cell cycle. H4K20me3 remains more stable and shows less dramatic changes compared to H4K20me1. H4K20me3 enhances chromatin folding and plays a structural role in the chromatin framework. Proper regulation of H4K20 methylation is vital for maintaining chromatin structure and function, as disruptions in this regulation result in genomic instability and other cellular dysfunctions.

References
[1] Jørgensen S, et al. Nucleic Acids Res. 2013;41(5):2797-2806.

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%