EZH1 Antibody [G17M2] | Primary Antibodies

Application: Reactivity: Human, Monkey

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

Reactivity
Human, Monkey

Source
Rabbit Monoclonal Antibody

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
95 kDa

Positive Control	K-562 cells; NCCIT cells; 293T cells; THP-1 cells; JK1 cells; Daudi cells; DU145 cells; HCT 116 cells
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. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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: 10%. 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. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 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 1. 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.

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.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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: 10%. 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. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 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

1. 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
EZH1 (D7D5D) Rabbit mAb detects endogenous levels of total EZH1 protein.

Subcellular Location
Nucleus

Uniprot ID
Q92800

Clone
G17M2

Synonym(s)
Histone-lysine N-methyltransferase EZH1; ENX-2; Enhancer of zeste homolog 1; EZH1; KIAA0388

Background
EZH1, or Enhancer of Zeste Homolog 1, is a Polycomb Group protein and catalytic subunit of the Polycomb Repressive Complex 2, working alongside SUZ12 and EED to methylate histone H3 at lysine 27, thereby enforcing transcriptional repression of developmental genes. Comprising about 747 amino acids, EZH1 contains a SET domain responsible for its methyltransferase activity, SANT1L and MCSS-SANT2L loop motifs that enhance nucleosome and DNA binding, and a unique EZH1/2 loop critical for substrate recognition and catalysis, which allows it to form either monomeric or dimeric PRC2:EZH1 assemblies with flexible conformations for chromatin interactions. EZH1 promotes the deposition of H3K27 trimethylation at silenced loci, leading to chromatin compaction, particularly effective when in dimeric form, maintaining heterochromatin, and repressing genes involved in cell identity, embryonic stem cell self-renewal, hematopoietic stem cell quiescence, prevention of senescence, myogenic differentiation, liver regeneration, and neuronal progenitor regulation. Although EZH1 has a lower catalytic rate than EZH2, it compensates by binding more stably to nucleosomes, supporting PRC2’s function in lineage commitment and homeostasis across tissues such as muscle, liver, and blood. EZH1 variants can disrupt neurogenesis and cause neurodevelopmental disorders, and combined inhibition of both EZH1 and EZH2 is more effective than targeting EZH2 alone in cancers like myeloma, lymphoma, and leukemia.

Background

EZH1, or Enhancer of Zeste Homolog 1, is a Polycomb Group protein and catalytic subunit of the Polycomb Repressive Complex 2, working alongside SUZ12 and EED to methylate histone H3 at lysine 27, thereby enforcing transcriptional repression of developmental genes. Comprising about 747 amino acids, EZH1 contains a SET domain responsible for its methyltransferase activity, SANT1L and MCSS-SANT2L loop motifs that enhance nucleosome and DNA binding, and a unique EZH1/2 loop critical for substrate recognition and catalysis, which allows it to form either monomeric or dimeric PRC2:EZH1 assemblies with flexible conformations for chromatin interactions. EZH1 promotes the deposition of H3K27 trimethylation at silenced loci, leading to chromatin compaction, particularly effective when in dimeric form, maintaining heterochromatin, and repressing genes involved in cell identity, embryonic stem cell self-renewal, hematopoietic stem cell quiescence, prevention of senescence, myogenic differentiation, liver regeneration, and neuronal progenitor regulation. Although EZH1 has a lower catalytic rate than EZH2, it compensates by binding more stably to nucleosomes, supporting PRC2’s function in lineage commitment and homeostasis across tissues such as muscle, liver, and blood. EZH1 variants can disrupt neurogenesis and cause neurodevelopmental disorders, and combined inhibition of both EZH1 and EZH2 is more effective than targeting EZH2 alone in cancers like myeloma, lymphoma, and leukemia.

References
https://pubmed.ncbi.nlm.nih.gov/33514705/ https://pubmed.ncbi.nlm.nih.gov/36476271/

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

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Handling Instructions

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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%