MLX Antibody [N17K22] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat, Monkey

Lane 1: K562, Lane 2: HCT116, Lane 3: COS-7

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:100 CHIP1:100

Application
WB, IP, ChIP

Reactivity
Human, Mouse, Rat, 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
30 kDa

Positive Control	MEF cells; K-562 cells; HCT-116 cells; COS7 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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 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
MLX Antibody (Rabbit mAb) [N17K22] detects endogenous levels of total MLX protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q9UH92

Clone
N17K22

Synonym(s)
Max-like protein X, Class D basic helix-loop-helix protein 13 (bHLHd13), Max-like bHLHZip protein, Protein BigMax, Transcription factor-like protein 4, MLX, BHLHD13, TCFL4

Background
MLX, a basic helix-loop-helix leucine zipper transcription factor in the Myc/Max/Mad superfamily, heterodimerizes obligatorily with tissue-specific partners MLXIP (MondoA) or MLXIPL (ChREBP) to sense glucose metabolites and regulate metabolic gene expression. Both complexes feature bHLHZip domains for E-box (CACGTG) DNA binding and N-terminal activation domains responsive to glucose-6-phosphate via hexokinase-catalyzed phosphorylation, with cytoplasmic-mitochondrial retention in low glucose shifting to nuclear accumulation upon metabolite elevation. MLX/MLXIP heterodimers translocate rapidly to the nucleus following 2-deoxyglucose exposure, binding ChoRE promoters of thioredoxin-interacting protein (TXNIP), ARRDC4, and glycolytic enzymes like HK2, PFKFB3, LDH-A to drive >75% of glucose-induced transcription, while promoting histone H4 acetylation for open chromatin. MLX/MLXIPL similarly activates lipogenic targets in the liver, with MLX phosphorylation stabilizing heterotetramers on DNA for sustained activity. Glucose sensing integrates hexokinase activity, as inhibitors like 3-bromopyruvate block nuclear import, and MondoA mutations disrupting MLX dimerization abolish response. MLX orchestrates glucose homeostasis by inducing glycolysis/lipogenesis genes and repressing uptake via TXNIP, balancing nutrient flux in muscle, liver, and proliferating cells. MLX-null impairs myokine secretion, including IGF2 for myoblast fusion/Akt activation, blunts muscle regeneration, and disrupts sugar tolerance/lipid balance. Dysregulation contributes to diabetes through failed TXNIP induction and cancer via Myc cooperation, reprogramming metabolism.

Background

MLX, a basic helix-loop-helix leucine zipper transcription factor in the Myc/Max/Mad superfamily, heterodimerizes obligatorily with tissue-specific partners MLXIP (MondoA) or MLXIPL (ChREBP) to sense glucose metabolites and regulate metabolic gene expression. Both complexes feature bHLHZip domains for E-box (CACGTG) DNA binding and N-terminal activation domains responsive to glucose-6-phosphate via hexokinase-catalyzed phosphorylation, with cytoplasmic-mitochondrial retention in low glucose shifting to nuclear accumulation upon metabolite elevation. MLX/MLXIP heterodimers translocate rapidly to the nucleus following 2-deoxyglucose exposure, binding ChoRE promoters of thioredoxin-interacting protein (TXNIP), ARRDC4, and glycolytic enzymes like HK2, PFKFB3, LDH-A to drive >75% of glucose-induced transcription, while promoting histone H4 acetylation for open chromatin. MLX/MLXIPL similarly activates lipogenic targets in the liver, with MLX phosphorylation stabilizing heterotetramers on DNA for sustained activity. Glucose sensing integrates hexokinase activity, as inhibitors like 3-bromopyruvate block nuclear import, and MondoA mutations disrupting MLX dimerization abolish response. MLX orchestrates glucose homeostasis by inducing glycolysis/lipogenesis genes and repressing uptake via TXNIP, balancing nutrient flux in muscle, liver, and proliferating cells. MLX-null impairs myokine secretion, including IGF2 for myoblast fusion/Akt activation, blunts muscle regeneration, and disrupts sugar tolerance/lipid balance. Dysregulation contributes to diabetes through failed TXNIP induction and cancer via Myc cooperation, reprogramming metabolism.

References
https://pubmed.ncbi.nlm.nih.gov/26584623/ https://pubmed.ncbi.nlm.nih.gov/18458340/

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%

MLX Antibody (Rabbit mAb) [N17K22]