MYH6+Slow Skeletal Myosin Heavy chain Antibody (Rabbit mAb) [E13G18]

Application: Reactivity: Mouse, Rat, Human

Lane 1: Mouse heart , Lane 2: Rat heart

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.

Usage Information

Dilution
WB1:500 IHC1:1000 - 1:2000

Application
WB, IHC

Reactivity
Mouse, Rat, Human

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
224 kDa

Positive Control	Human skeletal muscle tissue; Mouse heart tissue; Rat heart tissue
Negative Control	Human adult heart tissue

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: 250 mA, 180 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 1:500), 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: 250 mA, 180 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 1:500), 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
MYH6+Slow Skeletal Myosin Heavy chain Antibody (Rabbit mAb) [E13G18] detects endogenous levels of total MYH6+Slow Skeletal Myosin Heavy chain protein.

Subcellular Location
Cytoplasm, Thick filament

Uniprot ID
P13533

Clone
E13G18

Synonym(s)
MYHCA, MYH6, Myosin-6, Myosin heavy chain 6, MyHC-alpha, MYHCB, MYH7, Myosin-7, Myosin heavy chain 7, Myosin heavy chain slow isoform, MyHC-slow, MyHC-beta

Background
MYH6 encodes the cardiac α-myosin heavy chain, a sarcomeric motor protein of the myosin II family whose head domain hydrolyzes ATP and generates force along actin filaments, while slow skeletal myosin heavy chain isoforms share the same basic myosin architecture but are enriched in oxidative, fatigue-resistant skeletal fibers that support sustained, low-velocity contraction. The heavy chain polypeptide folds into an N-terminal motor domain containing the ATP-binding pocket and actin-interaction interface, followed by a neck region that binds regulatory and essential light chains and a long coiled-coil tail formed by a repetitive heptad pattern that dimerizes to build the thick filament backbone; limited proteolysis yields globular S1 heads and an S2/LMM rod segment, reflecting these functional and structural subdivisions. In atrial myocardium and conduction tissue, α-myosin heavy chain contributes to rapid cross-bridge cycling and efficient atrial contraction, and the MYH6 locus also embeds an intronic microRNA (miR-208a) that regulates expression of other myosin isoforms and conduction-related genes, so MYH6 products influence both mechanical performance and gene networks that shape the electrical phenotype of the atria. MYH6 is a central sarcomeric disease gene for familial atrial septal defects and a strong susceptibility locus for sick sinus syndrome, with rare and common variants in the α-myosin heavy chain altering sinus node function and atrial conduction; a missense variant p.Arg721Trp in the converter domain associates with a markedly increased lifetime risk of sick sinus syndrome and need for pacemaker implantation, consistent with the converter’s role in transmitting conformational changes from the motor domain to the coiled-coil tail during the power stroke. Additional MYH6 mutations, including in-frame deletions within the coiled-coil region that perturbs binding to myosin-binding protein C, impair sarcomere organization and slow conduction in atrial cardiomyocytes, and knockdown of myh6 in zebrafish reduces heart rate in a manner that can be rescued by wild-type but not mutant human α-myosin, indicating that a structurally intact MYH6 heavy chain is required for normal atrial excitation–contraction coupling and sinus node output. Across cardiac and slow skeletal muscle, α- and slow myosin heavy chains act as ATP-driven motors embedded in highly ordered thick filaments, with their head and converter domains setting contraction kinetics and their coiled-coil tails providing filament assembly interfaces, and MYH6 variants that disturb these domains or embedded regulatory microRNAs contribute to structural heart disease, atrial septal malformations, and primary conduction disorders such as sick sinus syndrome.

Background

MYH6 encodes the cardiac α-myosin heavy chain, a sarcomeric motor protein of the myosin II family whose head domain hydrolyzes ATP and generates force along actin filaments, while slow skeletal myosin heavy chain isoforms share the same basic myosin architecture but are enriched in oxidative, fatigue-resistant skeletal fibers that support sustained, low-velocity contraction. The heavy chain polypeptide folds into an N-terminal motor domain containing the ATP-binding pocket and actin-interaction interface, followed by a neck region that binds regulatory and essential light chains and a long coiled-coil tail formed by a repetitive heptad pattern that dimerizes to build the thick filament backbone; limited proteolysis yields globular S1 heads and an S2/LMM rod segment, reflecting these functional and structural subdivisions. In atrial myocardium and conduction tissue, α-myosin heavy chain contributes to rapid cross-bridge cycling and efficient atrial contraction, and the MYH6 locus also embeds an intronic microRNA (miR-208a) that regulates expression of other myosin isoforms and conduction-related genes, so MYH6 products influence both mechanical performance and gene networks that shape the electrical phenotype of the atria. MYH6 is a central sarcomeric disease gene for familial atrial septal defects and a strong susceptibility locus for sick sinus syndrome, with rare and common variants in the α-myosin heavy chain altering sinus node function and atrial conduction; a missense variant p.Arg721Trp in the converter domain associates with a markedly increased lifetime risk of sick sinus syndrome and need for pacemaker implantation, consistent with the converter’s role in transmitting conformational changes from the motor domain to the coiled-coil tail during the power stroke. Additional MYH6 mutations, including in-frame deletions within the coiled-coil region that perturbs binding to myosin-binding protein C, impair sarcomere organization and slow conduction in atrial cardiomyocytes, and knockdown of myh6 in zebrafish reduces heart rate in a manner that can be rescued by wild-type but not mutant human α-myosin, indicating that a structurally intact MYH6 heavy chain is required for normal atrial excitation–contraction coupling and sinus node output. Across cardiac and slow skeletal muscle, α- and slow myosin heavy chains act as ATP-driven motors embedded in highly ordered thick filaments, with their head and converter domains setting contraction kinetics and their coiled-coil tails providing filament assembly interfaces, and MYH6 variants that disturb these domains or embedded regulatory microRNAs contribute to structural heart disease, atrial septal malformations, and primary conduction disorders such as sick sinus syndrome.

References
https://pubmed.ncbi.nlm.nih.gov/19922871/ https://pubmed.ncbi.nlm.nih.gov/21378987/

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%