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Anti-c-Fos Rabbit Antibody [M9L15]

Catalog No.: F0267

Application: Reactivity: Human, Mouse, Rat

Lane 1: HeLa (serum-starved overnight; TPA,4 hours)
Lane 2: HeLa (serum-starved overnight)

Customer Review

Experiment Essentials

WB
The endogenous level of c-Fos in certain cell lines such as Hela is relatively low. It is recommended to add inducing stimulation (such as overnight serum starvation treatment and TPA treatment for another 4 hours) to maintain a high endogenous level of c-Fos.
After stimulation, the expression of c-Fos is rapid and short-lived. It is recommended to collect samples immediately after the stimulation is completed to prevent protein degradation.

Usage Information

Dilution
WB1:1000 FCM1:1600 - 1:6400 CHIP1:50

Application
WB, FCM, ChIP

Reactivity
Human, Mouse, Rat

Source
Rabbit

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
62 kDa

Positive Control	mouse cerebral cortex; mouse pons; Hela (serum-starved overnight + 400nM TPA-stimulated, 4 h); H-4-II-E (serum-starved overnight + 400nM TPA-stimulated, 4 h); NIH/3T3 (serum-starved overnight + 400nM TPA-stimulated, 4 h); PC-12 (serum-starved overnight, treated with h-βNGF, 50ng/ml, 2h)
Negative Control	Hela; H-4-II-E; NIH/3T3; PC-12

Expression & Treatment Conditions

Sample	Treatment Conditions
Hela	Serum-starvation (overnight) + TPA (400nM, 4 h)
H-4-II-E	Serum-starvation (overnight) + TPA (400nM, 4 h)
NIH/3T3	Serum-starvation (overnight) + TPA (400nM, 4 h)
NIH/3T3	Serum-starvation (overnight) + TPA (400nM, 4 h) + λ-phosphatase
PC-12	Serum-starvation (overnight) + h-βNGF (50ng/mL, 2h)
Click to view more sample data

^*For predicted expression levels of this protein in various human-derived cells and tissues, please refer to: http://www.proteinatlas.org

Sample	Treatment Conditions

Exprimental 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 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/NP-40 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/NP-40 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 928. 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/NP-40 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/NP-40 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/NP-40 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

928. 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
Anti-c-Fos Rabbit Antibody [M9L15] detects endogenous levels of total c-Fos protein.

Subcellular Location
Cytoplasm, Endoplasmic reticulum, Nucleus

Uniprot ID
P01100

Clone
M9L15

Background
The Fos family of nuclear oncogenes consists of c-Fos, FosB, Fos-related antigen 1 (FRA1), and Fos-related antigen 2 (FRA2). Unlike other Fos proteins, which exist as a single isoform, FosB has two variants: the full-length FosB and a truncated version known as FosB2 (Delta FosB), which lacks the carboxy-terminal 101 amino acids. Fos proteins are rapidly and transiently expressed in response to various extracellular signals such as growth factors, cytokines, neurotransmitters, polypeptide hormones, and stress. These proteins dimerize with Jun family members (c-Jun, JunB, and JunD) to form the transcription factor Activator Protein-1 (AP-1), which binds to TRE/AP-1 elements in DNA to regulate gene transcription. Fos and Jun proteins have a leucine-zipper motif essential for dimerization and a basic domain that facilitates DNA binding. The different Fos/Jun heterodimers have varying capacities to activate AP-1 dependent genes. Beyond expression levels, phosphorylation of Fos proteins by Erk kinases in response to external stimuli can enhance their transcriptional activity. Specifically, phosphorylation of c-Fos at Ser32 and Thr232 by Erk5 increases its stability and nuclear localization. Abnormal expression of c-Fos, FosB, or FRA2 can lead to neoplastic transformation of cells.

Background

The Fos family of nuclear oncogenes consists of c-Fos, FosB, Fos-related antigen 1 (FRA1), and Fos-related antigen 2 (FRA2). Unlike other Fos proteins, which exist as a single isoform, FosB has two variants: the full-length FosB and a truncated version known as FosB2 (Delta FosB), which lacks the carboxy-terminal 101 amino acids. Fos proteins are rapidly and transiently expressed in response to various extracellular signals such as growth factors, cytokines, neurotransmitters, polypeptide hormones, and stress. These proteins dimerize with Jun family members (c-Jun, JunB, and JunD) to form the transcription factor Activator Protein-1 (AP-1), which binds to TRE/AP-1 elements in DNA to regulate gene transcription. Fos and Jun proteins have a leucine-zipper motif essential for dimerization and a basic domain that facilitates DNA binding. The different Fos/Jun heterodimers have varying capacities to activate AP-1 dependent genes. Beyond expression levels, phosphorylation of Fos proteins by Erk kinases in response to external stimuli can enhance their transcriptional activity. Specifically, phosphorylation of c-Fos at Ser32 and Thr232 by Erk5 increases its stability and nuclear localization. Abnormal expression of c-Fos, FosB, or FRA2 can lead to neoplastic transformation of cells.

References
https://pubmed.ncbi.nlm.nih.gov/10963134/ https://pubmed.ncbi.nlm.nih.gov/17018293/

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%