SREBP1 Antibody (Rabbit mAb) [J4J22]

Application: Reactivity: Human, Mouse

Lane 1: HCT 116, Lane 2: A549, Lane 3: Huh7

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse

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 Observed MW
122 kDa 125 kDa, 87 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human adrenal gland tissue; Mouse liver tissue; 3T3-L1 cells (differentiated for 6 days); HeLa cells; HCT 116 cells; Huh7 cells; A549 cells; HT-29 cells; Hepa1-6 cells
Negative Control	3T3-L1 cells

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 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/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. Lyse the cells by adding 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: 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: 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 primary antibody dilution buffer 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 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/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. Lyse the cells by adding 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: 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: 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 primary antibody dilution buffer 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
SREBP1 Antibody (Rabbit mAb) [J4J22] detects endogenous levels of total SREBP1 protein.

Subcellular Location
Cytoplasmic vesicle, Endoplasmic reticulum, Golgi apparatus, Membrane, Nucleus

Uniprot ID
P36956

Clone
J4J22

Synonym(s)
Srebp1, Srebf1, Sterol regulatory element-binding protein 1, SREBP-1, Sterol regulatory element-binding transcription factor 1

Background
SREBP-1 belongs to the SREBP family of membrane-bound basic helix-loop-helix leucine zipper transcription factors and is synthesized as an inactive precursor embedded in the endoplasmic reticulum membrane, existing as two isoforms, SREBP-1A and SREBP-1C, generated from alternative transcription start sites; SREBP-1A is expressed selectively in a limited set of tissues and shows stronger transcriptional activity capable of driving both lipogenic and cholesterogenic gene programs, while SREBP-1C is the predominant isoform across most tissues, carries weaker transcriptional activity, and primarily controls lipogenic gene expression. The precursor associates in the ER membrane with SCAP, SREBP cleavage-activating protein, which functions as the cholesterol-sensing component of this regulatory system, and under sterol-replete conditions, SCAP binds the ER-resident protein INSIG-1 through SCAP's sterol-sensing domain, and this SCAP-INSIG interaction physically retains the SCAP-SREBP complex within the ER by preventing recognition of a cytosolic transport signal by COPII coat proteins, blocking the complex from entering ER-to-Golgi transport vesicles. When cellular sterol levels fall, sterol-induced SCAP-INSIG binding is lost, freeing the SCAP-SREBP complex to exit the ER and travel to the Golgi apparatus, where SREBP-1 undergoes a two-step, obligatorily sequential proteolytic activation: site-1 protease first cleaves SREBP-1 within its luminal loop, generating an intermediate in which the N-terminal transcription factor domain remains membrane-anchored, and site-2 protease then cleaves this intermediate, releasing the transcription factor domain from the membrane entirely; site-2 protease cannot act on full-length SREBP-1 if site-1 cleavage has not already occurred, making the two proteolytic steps strictly ordered rather than independently triggerable. The released N-terminal transcription factor fragment translocates to the nucleus, where it binds target gene promoters through dual sequence specificity, recognizing both the canonical sterol regulatory element SRE-1 and an E-box motif, and drives transcription of genes governing cholesterol biosynthesis, fatty acid synthesis, and the LDL receptor pathway. Because SCAP escorts SREBP-1 through this ER-to-Golgi transport step and is required for site-1 protease access, SCAP functions as the rate-limiting, sterol-regulated checkpoint of the entire pathway, and liver-specific deletion of SCAP eliminates processing of all SREBP isoforms and drastically reduces hepatic lipid production, directly establishing SCAP as necessary for SREBP-1 activation in vivo. SREBP-1C additionally strongly activates global lipid synthesis specifically in rapidly proliferating cells and contributes to the innate immune response in macrophages through regulation of lipid metabolism, and dysregulated SREBP-1 processing is implicated in metabolic liver disease.

Background

SREBP-1 belongs to the SREBP family of membrane-bound basic helix-loop-helix leucine zipper transcription factors and is synthesized as an inactive precursor embedded in the endoplasmic reticulum membrane, existing as two isoforms, SREBP-1A and SREBP-1C, generated from alternative transcription start sites; SREBP-1A is expressed selectively in a limited set of tissues and shows stronger transcriptional activity capable of driving both lipogenic and cholesterogenic gene programs, while SREBP-1C is the predominant isoform across most tissues, carries weaker transcriptional activity, and primarily controls lipogenic gene expression. The precursor associates in the ER membrane with SCAP, SREBP cleavage-activating protein, which functions as the cholesterol-sensing component of this regulatory system, and under sterol-replete conditions, SCAP binds the ER-resident protein INSIG-1 through SCAP's sterol-sensing domain, and this SCAP-INSIG interaction physically retains the SCAP-SREBP complex within the ER by preventing recognition of a cytosolic transport signal by COPII coat proteins, blocking the complex from entering ER-to-Golgi transport vesicles. When cellular sterol levels fall, sterol-induced SCAP-INSIG binding is lost, freeing the SCAP-SREBP complex to exit the ER and travel to the Golgi apparatus, where SREBP-1 undergoes a two-step, obligatorily sequential proteolytic activation: site-1 protease first cleaves SREBP-1 within its luminal loop, generating an intermediate in which the N-terminal transcription factor domain remains membrane-anchored, and site-2 protease then cleaves this intermediate, releasing the transcription factor domain from the membrane entirely; site-2 protease cannot act on full-length SREBP-1 if site-1 cleavage has not already occurred, making the two proteolytic steps strictly ordered rather than independently triggerable. The released N-terminal transcription factor fragment translocates to the nucleus, where it binds target gene promoters through dual sequence specificity, recognizing both the canonical sterol regulatory element SRE-1 and an E-box motif, and drives transcription of genes governing cholesterol biosynthesis, fatty acid synthesis, and the LDL receptor pathway. Because SCAP escorts SREBP-1 through this ER-to-Golgi transport step and is required for site-1 protease access, SCAP functions as the rate-limiting, sterol-regulated checkpoint of the entire pathway, and liver-specific deletion of SCAP eliminates processing of all SREBP isoforms and drastically reduces hepatic lipid production, directly establishing SCAP as necessary for SREBP-1 activation in vivo. SREBP-1C additionally strongly activates global lipid synthesis specifically in rapidly proliferating cells and contributes to the innate immune response in macrophages through regulation of lipid metabolism, and dysregulated SREBP-1 processing is implicated in metabolic liver disease.

References
https://pubmed.ncbi.nlm.nih.gov/12202038/ https://pubmed.ncbi.nlm.nih.gov/32385422/

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%