MAFA Antibody [N15J3] | Primary Antibodies

Application: Reactivity: Human, Mouse

Lane 1: 293T, Lane 2: 293T (hMAFA transfected)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IP1:200 IF1:1000 CHIP1:100

Application
WB, IP, IF, ChIP

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
37 kDa 195 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	Adult mouse pancreas; β-TC-6 cells; NIT-1 cells
Negative Control	C2C12 cells; NIH/3T3 cells

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, 60 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/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, 60 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
MAFA Antibody (Rabbit mAb) [N15J3] detects endogenous levels of total MAFA protein.

Subcellular Location
Nucleus

Uniprot ID
Q8NHW3

Clone
N15J3

Synonym(s)
hMafA; INSDM; MAF bZIP transcription factor A; MAFA; v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian)

Background
MAFA, a member of the large MAF basic leucine-zipper transcription factor family, functions as a β cell–enriched regulator that confers tissue specificity and glucose responsiveness on the insulin gene and a broader set of β cell genes involved in stimulus–secretion coupling and hormone processing. The protein contains an N‑terminal transactivation domain that integrates glucose- and kinase-dependent regulatory inputs and a C‑terminal basic leucine-zipper region that mediates dimerization and binding to Maf recognition elements within cis-regulatory regions such as the C1/RIPE3b element of the insulin promoter, where MAFA acts in concert with PDX1 and NEUROD1 to drive β cell–selective transcription. Glucose regulates MAFA at both transcriptional and post-translational levels, with elevated glucose increasing MAFA expression and DNA-binding activity and with constitutive phosphorylation of multiple N‑terminal serine and threonine residues by glycogen synthase kinase 3 modulating protein stability so that changes in ambient glucose alter MAFA turnover and, in turn, insulin promoter occupancy. MAFA directly activates the insulin gene and also controls a network of β-cell genes central to insulin biosynthesis, metabolism-secretion coupling, and incretin responsiveness, including GLUT2, glucokinase, PDX1, NKX6.1, GLP1R, PCSK1, and pyruvate carboxylase, thereby coordinating glucose uptake, metabolic flux, proinsulin processing, and secretory capacity under physiological conditions. Expression of MAFA is largely restricted to insulin-producing β cells within adult islets, and the appearance of MAFA during the second wave of β cell differentiation marks the acquisition of mature β cell identity and robust insulin secretory function, while reduced MAFA levels accompany chronic hyperglycemia and β cell dysfunction in diabetic states. MAFA overexpression enhances insulin transcription, insulin content, and glucose-stimulated insulin secretion, whereas dominant-negative MAFA dampens these outputs and downregulates key β cell genes, indicating that MAFA sits near the top of a transcriptional hierarchy that maintains the mature β cell phenotype and preserves secretory performance under metabolic load. MAFA also participates in β cell reprogramming strategies, where its coexpression with PDX1 and NEUROD1 efficiently induces insulin biosynthesis in non–β cells and promotes conversion of islet α cells and progenitors toward a β cell–like state, linking this factor mechanistically to lineage specification and plasticity within the islet endocrine compartment. Germline or functional alterations in MAFA and its regulatory phosphosites associate with impaired insulin gene expression, β cell failure, and monogenic diabetes phenotypes, and MAFA downregulation is a consistent molecular feature of human type 2 diabetic islets.

Background

MAFA, a member of the large MAF basic leucine-zipper transcription factor family, functions as a β cell–enriched regulator that confers tissue specificity and glucose responsiveness on the insulin gene and a broader set of β cell genes involved in stimulus–secretion coupling and hormone processing. The protein contains an N‑terminal transactivation domain that integrates glucose- and kinase-dependent regulatory inputs and a C‑terminal basic leucine-zipper region that mediates dimerization and binding to Maf recognition elements within cis-regulatory regions such as the C1/RIPE3b element of the insulin promoter, where MAFA acts in concert with PDX1 and NEUROD1 to drive β cell–selective transcription. Glucose regulates MAFA at both transcriptional and post-translational levels, with elevated glucose increasing MAFA expression and DNA-binding activity and with constitutive phosphorylation of multiple N‑terminal serine and threonine residues by glycogen synthase kinase 3 modulating protein stability so that changes in ambient glucose alter MAFA turnover and, in turn, insulin promoter occupancy. MAFA directly activates the insulin gene and also controls a network of β-cell genes central to insulin biosynthesis, metabolism-secretion coupling, and incretin responsiveness, including GLUT2, glucokinase, PDX1, NKX6.1, GLP1R, PCSK1, and pyruvate carboxylase, thereby coordinating glucose uptake, metabolic flux, proinsulin processing, and secretory capacity under physiological conditions. Expression of MAFA is largely restricted to insulin-producing β cells within adult islets, and the appearance of MAFA during the second wave of β cell differentiation marks the acquisition of mature β cell identity and robust insulin secretory function, while reduced MAFA levels accompany chronic hyperglycemia and β cell dysfunction in diabetic states. MAFA overexpression enhances insulin transcription, insulin content, and glucose-stimulated insulin secretion, whereas dominant-negative MAFA dampens these outputs and downregulates key β cell genes, indicating that MAFA sits near the top of a transcriptional hierarchy that maintains the mature β cell phenotype and preserves secretory performance under metabolic load. MAFA also participates in β cell reprogramming strategies, where its coexpression with PDX1 and NEUROD1 efficiently induces insulin biosynthesis in non–β cells and promotes conversion of islet α cells and progenitors toward a β cell–like state, linking this factor mechanistically to lineage specification and plasticity within the islet endocrine compartment. Germline or functional alterations in MAFA and its regulatory phosphosites associate with impaired insulin gene expression, β cell failure, and monogenic diabetes phenotypes, and MAFA downregulation is a consistent molecular feature of human type 2 diabetic islets.

References
https://pubmed.ncbi.nlm.nih.gov/17149590/ https://pubmed.ncbi.nlm.nih.gov/12368292/

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%

MAFA Antibody (Rabbit mAb) [N15J3]