Elk-1 Antibody [J24L11] | Primary Antibodies

Application: Reactivity: Human, Monkey

Lane 1: SNU-1, Lane 2: KATI Ⅲ

Usage Information

Dilution
WB1:1000 IP1:200 CHIP1:50

Application
WB, IP, ChIP

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
65 kDa

Positive Control	SNU-1 cells; KATO III cells; 786-O cells; HeLa cells; 293T 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/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 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), 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. Lyse the cells by adding 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: 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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), 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. Lyse the cells by adding 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: 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
Elk-1 Antibody (Rabbit mAb) [J24L11] detects endogenous levels of total Elk-1 protein.

Subcellular Location
Nucleus

Uniprot ID
P19419

Clone
J24L11

Synonym(s)
ETS domain-containing protein Elk-1; ELK1

Background
Elk‑1 is a member of the ternary complex factor (TCF) subfamily of ETS‑domain transcription factors that partners with serum response factor (SRF) at serum response elements (SREs) to control immediate‑early and growth‑responsive gene expression in response to serum, growth factors, and stress. Elk‑1 contains an N‑terminal ETS DNA‑binding domain, a central SRF‑interacting B‑box region, and a C‑terminal transcriptional activation domain (TAD) harboring a cluster of serine‑ and threonine‑rich motifs whose phosphorylation by MAPKs and other kinases regulates Elk‑1 activity, with Ser383 representing a key regulatory site. Elk‑1 is directly phosphorylated by ERK, p38, and stress‑activated JNK at the TAD, and ERK‑dependent phosphorylation of Ser383 and adjacent residues not only increases Elk‑1 binding to the Elk‑1–SRF–SRE ternary complex but also promotes recruitment of coactivators such as p300/CBP and the Mediator subunit MED23, thereby stimulating histone acetylation, Mediator‑dependent pre‑initiation‑complex assembly, and rapid induction of SRE‑driven genes such as c‑fos and Egr1. Mitogen‑dependent phosphorylation of Elk‑1 drives nuclear translocation, SRE‑linked chromatin remodeling, and transcriptional programs tied to synaptic activity and plasticity, whereas sumoylation‑dependent cytoplasmic sequestration and multisite phosphorylation by SAPKs like JNK tune the balance between activation and repression, and Elk‑1 deregulation has been implicated in proliferative signaling, stress responses, and neurodegenerative and oncogenic contexts.

Background

Elk‑1 is a member of the ternary complex factor (TCF) subfamily of ETS‑domain transcription factors that partners with serum response factor (SRF) at serum response elements (SREs) to control immediate‑early and growth‑responsive gene expression in response to serum, growth factors, and stress. Elk‑1 contains an N‑terminal ETS DNA‑binding domain, a central SRF‑interacting B‑box region, and a C‑terminal transcriptional activation domain (TAD) harboring a cluster of serine‑ and threonine‑rich motifs whose phosphorylation by MAPKs and other kinases regulates Elk‑1 activity, with Ser383 representing a key regulatory site. Elk‑1 is directly phosphorylated by ERK, p38, and stress‑activated JNK at the TAD, and ERK‑dependent phosphorylation of Ser383 and adjacent residues not only increases Elk‑1 binding to the Elk‑1–SRF–SRE ternary complex but also promotes recruitment of coactivators such as p300/CBP and the Mediator subunit MED23, thereby stimulating histone acetylation, Mediator‑dependent pre‑initiation‑complex assembly, and rapid induction of SRE‑driven genes such as c‑fos and Egr1. Mitogen‑dependent phosphorylation of Elk‑1 drives nuclear translocation, SRE‑linked chromatin remodeling, and transcriptional programs tied to synaptic activity and plasticity, whereas sumoylation‑dependent cytoplasmic sequestration and multisite phosphorylation by SAPKs like JNK tune the balance between activation and repression, and Elk‑1 deregulation has been implicated in proliferative signaling, stress responses, and neurodegenerative and oncogenic contexts.

References
https://pubmed.ncbi.nlm.nih.gov/12514134/ https://pubmed.ncbi.nlm.nih.gov/21441990/

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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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%

Elk-1 Antibody (Rabbit mAb) [J24L11]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support