SET1A Antibody [K11A18] | Primary Antibodies

Application: Reactivity: Human

Lane 1: 293T, Lane 2: Hela

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.

Usage Information

Dilution
WB1:1000 IP1:200 CHIP1:100

Application
WB, IP, ChIP

Reactivity
Human

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
300 kDa

Positive Control	293T cells; HeLa cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 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/NP-40 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/NP-40 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: 5%. 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: 250 mA, 180 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/NP-40 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/NP-40 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/NP-40 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: 5%. 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: 250 mA, 180 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
SET1A Antibody [K11A18] detects endogenous levels of total SET1A protein.

Subcellular Location
Chromosome, Cytoplasm, Nucleus

Uniprot ID
O15047

Clone
K11A18

Synonym(s)
Histone-lysine N-methyltransferase SETD1A; Lysine N-methyltransferase 2F; SET domain-containing protein 1A (hSET1A); Set1/Ash2 histone methyltransferase complex subunit SET1; SETD1A; KIAA0339; KMT2F; SET1; SET1A

Background
SET1A, also known as SETD1A or KMT2F, is a mammalian homolog of yeast Set1 and serves as the catalytic subunit of the SET1A/COMPASS histone H3 lysine 4 methyltransferase complex, which is essential for embryonic stem cell self-renewal, early development, and differentiation. SET1A has a conserved C-terminal SET domain that is crucial for its methyltransferase activity, primarily generating H3K4 di- and trimethylation, and is flanked by n-SET and post-SET domains, as well as regions that enable interactions with core subunits such as WDR5, RBBP5, ASH2L, and DPY30, collectively known as the WRAD complex, which greatly enhances its activity. It also partners with unique proteins like CFP1 for CpG island binding and WDR82 for linking to the RNA polymerase II C-terminal domain. SET1A’s main role is to deposit H3K4 trimethylation at promoters of active or poised genes, promoting an open chromatin state and transcriptional activation. Notably, while the SET domain is not required for embryonic stem cell proliferation or maintenance of pluripotency markers such as Nanog, Oct4, and Sox2 due to its non-catalytic scaffolding functions, it is indispensable for proper differentiation by driving H3K4 trimethylation at developmental genes including Bmp5, Epor, the HoxA gene cluster, and bivalent loci, supporting mesoderm specification, embryoid body formation, and preventing the abnormal persistence of pluripotency factors. During development, SET1A collaborates with MLL2 for stage-specific H3K4 trimethylation switching, with MLL2 acting in self-renewal and SET1A in differentiation, regulates Hox gene expression, and non-enzymatically supports mitotic fidelity and DNA repair. Mutations or loss of SET1A result in embryonic lethality at early stages, are linked to neurodevelopmental disorders such as autism and schizophrenia due to defects in H3K4 trimethylation, and its overexpression in cancers can enhance proliferation and invasion through pathways involving YAP, Wnt, and HIF1 alpha.

Background

SET1A, also known as SETD1A or KMT2F, is a mammalian homolog of yeast Set1 and serves as the catalytic subunit of the SET1A/COMPASS histone H3 lysine 4 methyltransferase complex, which is essential for embryonic stem cell self-renewal, early development, and differentiation. SET1A has a conserved C-terminal SET domain that is crucial for its methyltransferase activity, primarily generating H3K4 di- and trimethylation, and is flanked by n-SET and post-SET domains, as well as regions that enable interactions with core subunits such as WDR5, RBBP5, ASH2L, and DPY30, collectively known as the WRAD complex, which greatly enhances its activity. It also partners with unique proteins like CFP1 for CpG island binding and WDR82 for linking to the RNA polymerase II C-terminal domain. SET1A’s main role is to deposit H3K4 trimethylation at promoters of active or poised genes, promoting an open chromatin state and transcriptional activation. Notably, while the SET domain is not required for embryonic stem cell proliferation or maintenance of pluripotency markers such as Nanog, Oct4, and Sox2 due to its non-catalytic scaffolding functions, it is indispensable for proper differentiation by driving H3K4 trimethylation at developmental genes including Bmp5, Epor, the HoxA gene cluster, and bivalent loci, supporting mesoderm specification, embryoid body formation, and preventing the abnormal persistence of pluripotency factors. During development, SET1A collaborates with MLL2 for stage-specific H3K4 trimethylation switching, with MLL2 acting in self-renewal and SET1A in differentiation, regulates Hox gene expression, and non-enzymatically supports mitotic fidelity and DNA repair. Mutations or loss of SET1A result in embryonic lethality at early stages, are linked to neurodevelopmental disorders such as autism and schizophrenia due to defects in H3K4 trimethylation, and its overexpression in cancers can enhance proliferation and invasion through pathways involving YAP, Wnt, and HIF1 alpha.

References
https://pubmed.ncbi.nlm.nih.gov/32795105/ https://pubmed.ncbi.nlm.nih.gov/34803610/

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

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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%