support data
| REACTIVITY | H.M.R |
| SENSITIVITY | endogenous |
| PM (kDa) | 230 |
| FUENTE | Coelho |
Application Key:
- WB-Western spot
- IP- Immunoprecipitation
- IHC- Immunhistochemie
- Switch- Immunoprecipitation of chromatin
- C&R-CUT AND RUN
- CONNECTICUT- CUTTING AND LABELING
- Database-Just-Point
- eCLIP-eCLIP
- E- Immunofluorescence
- F- Flow cytometry
Reactivity key between species:
- H-Human
- METRO-Maus
- R-Rata
- mmm-Hamster
- mk-Mono
- For-Virus
- Mi- mink
- C-Polo
- DM-D. Melanogaster
- X-Xenopus
- Z-zebrafish
- B-Bovine
- DG-puppy
- book page-Pork meat
- Caroline all on- Street. Beer
- Ce-W. elegant
- Hour-Horse
- family doctor-Guinea pig
- Rab-Coelho
- at-All expected species
- similar products
Product information
Product Usage Information
| Application | dilution |
|---|---|
| transmitted west | 1:1000 |
| Immunofluorescence (immunocytochemistry) | 1:50 |
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Supplied in 10mM Sodium HEPES (pH 7.5), 150mM NaCl, 100µg/mL BSA and 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Protocol
PRESS
Western blot protocol
For Western blots, incubate the membrane with primary antibody diluted in 5% w/v BSA, 1X TBS, 0.1% Tween.®20 to 4°C with gentle stirring overnight.
USE: Refer to the primary antibody product website for recommended antibody dilution.
A. Solutions and Reagents
From sample preparation to detection, the reagents you need for Western blotting are now in one convenient kit:#12957Solution kit for western blot applications
USE: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.
- 20X phosphate buffered saline (PBS): (#9808) To prepare 1 L of 1X PBS: add 50 mL of 20X PBS to 950 mL of dH2Hallo Mix.
- 10X Tris-buffered saline (TBS): (#12498) To prepare 1L of 1X TBS: add 100mL of 10X to 900mL of dH2Hallo Mix.
- 1X SDS-Probenpuffer: Blue Load Pack (#7722) or the red cargo package (#7723) Prepare a new 3X Reducer Loading Buffer by adding 1/10 volume 30X DTT to 1 volume 3X SDS Loading Buffer. Dilute 1X with dH2Ö
- 10x Tris-Glycin SDS Laufpuffer: (#4050) To prepare 1L of 1X Run Buffer: Add 100mL of 10X Run Buffer to 900mL of dH2Hallo Mix.
- 10X Tris-Glycin-Transferpuffer: (#12539) Preparation of 1 L of 1x Transfer Buffer: add 100 mL of 10x Transfer Buffer to 200 mL of methanol + 700 mL of dH2Hallo Mix.
- 10X Tris-buffered saline with interpolation®20 (TBST): (#9997) Preparation of 1L 1X TBST: Add 100mL 10X TBST to 900mL dH2Hallo Mix.
- Magermilchpulver: (#9999).
- Crash-Puffer: 1X TBST with 5% w/v skimmed milk powder; For 150ml add 7.5g skimmed milk powder to 150ml 1X TBST and mix well.
- wash buffer: (#9997) 1X TBS.
- Rinderserum albumin (BSA): (#9998).
- Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; For 20mL add 1.0g BSA to 20mL 1X TBST and mix well.
- Biotinylated protein ladder detection package: (#7727).
- Prestained blue protein marker, large range (11-250 kDa): (#59329).
- membrane and blotting paper: (#12369) This protocol has been optimized for nitrocellulose membranes. In general, a pore size of 0.2 µm is recommended.
- HRP conjugated secondary antibody: anti-rabbit IgG, antibody linked to HRP (#7074).
- detection reagent: Reaktives SignalFire™ ECL (#6883).
B. Proteintransfer
A general protocol for sample preparation.
- Treat the cells by adding fresh media with buffer for the desired time.
- Aspirate the culture medium; wash cells with 1X PBS; striving.
- Lyse the cells by adding 1x SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells from the plate and transfer the extract to a microcentrifuge tube. stay on ice
- Sonicate for 10-15 seconds to complete cell lysis and DNA cleavage (to reduce sample stickiness).
- Heat a 20 L sample at 95–100 °C for 5 min; cool on ice.
- Microcentrifuge for 5 min.
Cargue 20 l Gel SDS-PAGE (10 cm x 10 cm).
USE: loading of prestained molecular weight markers (#59329, 10 µl/lane) to check the electroblotting and the biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights.
- Electroblot on nitrocellulose membrane (#12369).
C. Membrane blocking and antibody incubations
USE: Volumes are for 10cm x 10cm (100cm2) membrane; Adjust volumes accordingly for different sized membranes.
I. Membranblock
- (Optional) After transfer, wash the nitrocellulose membrane with 25 mL of TBS for 5 min at room temperature.
- Incubate the membrane in 25 mL of blocking buffer for 1 hour at room temperature.
- Wash three times for 5 min each with 15 mL of TBST.
II. Primary Antibody Incubation
- Incubate the membrane and primary antibody (using the appropriate diluent and diluent as recommended on the product page) in 10 mL of Primary Antibody Dilution Buffer with gentle shaking overnight at 4°C.
- Wash three times for 5 min each with 15 mL of TBST.
- Incubate the membrane with anti-rabbit IgG, HRP-linked antibody (#7074to 1:2000) and anti-biotin, antibody linked to HRP (#7075a 1:1000–1:3000) for the detection of biotinylated protein markers in 10 ml of blocking buffer with gentle shaking for 1 hour at room temperature.
- Wash three times for 5 min each with 15 mL of TBST.
- Proceed with screening (Section D).
D. Proteinnachweis
Instructions for use:
- Wash the membrane-bound HRP (conjugated antibody) three times for 5 min in TBST.
- Prepare 1X reativo SignalFire™ ECL (#6883) by diluting one part Reagent A 2x and one part Reagent B 2x (e.g. for 10mL add 5mL Reagent A and 5mL Reagent B). Mix well.
- Incubate substrate with membrane for 1 minute, shake off excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
* Avoid repeated skin contact.
Released June 2005
reviewed in June 2020
Protocol identification: 10
Immunofluorescence (immunocytochemistry)
A. Solutions and Reagents
Get high-quality immunofluorescence images with efficient and inexpensive prepackaged reagents in our#12727Immunofluorescence application solution kit
USE: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.
- 20X phosphate buffered saline (PBS):(9808) To prepare 1 L of 1X PBS: add 50 mL of 20X PBS to 950 mL of dH2Hello Mix. Adjust the pH to 8.0.
- Formaldehyde: 16%, no methanol, Polysciences, Inc. (Cat# 18814), use new vials and store uncapped at 4°C in the dark, dilute in 1X PBS for use.
- Crash-Puffer(1X PBS / 5% normal serum / 0.3% Triton™ X-100): To make 10mL, add 0.5mL of normal serum of the same species as the secondary antibody (e.g. normal goat serum (#5425)) y 0,5 ml 20X PBS a 9,0 ml dH2Mix well. Add 30 µl of Triton™ X-100 while stirring.
- Antibody Dilution Buffer(1X PBS / 1% BSA / 0.3% Triton X-100): To make 10 mL, add 30 µL of Triton™ X-100 to 10 mL of 1X PBS. Mix well and add 0.1 g BSA (9998), Mix.
Recommended fluorochrome-conjugated anti-rabbit secondary antibodies:
- Rabbit-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® 488 Conjugate) # 4412
- Rabbit-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® 555 Conjugate) # 4413
- Kaninchen-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® Conjugate 594) # 8889
- Kaninchen-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® Conjugate 647) # 4414
- Expand®Gold-Antifade-Reagenz(#9071),Expand®Gold AntiFade Reagent with DAPI(#8961).
B. Sample preparation: cultured cell lines (IF-IC)
USE: Cells must be cultured, treated, fixed, and stained directly on multiwell plates, chamber slides, or coverslips.
-
Aspirate the liquid and then cover the cells to a depth of 2-3 mm with 4% formaldehyde diluted in 1X PBS.
USE: Formaldehyde is toxic, use only in a fume hood.
- Allow cells to sit at room temperature for 15 min.
- Aspirate fixative, rinse three times in 1X PBS for 5 min each.
- Proceed with immunostaining (section C).
C. Immunostaining
USE: All subsequent incubations, unless otherwise specified, should be performed at room temperature in a light-tight humid box or covered dish/dish to prevent drying and fading of the fluorochrome.
- Block the sample in blocking buffer for 60 min.
- During blocking, prepare the primary antibody by diluting it as directed on the product page under Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Incubate the sample in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1 to 2 hours at room temperature in the dark.
- Rinse three times in 1X PBS for 5 min each.
- Coverslips with prolong®Gold-Anti-Fade-Reagenz (#9071) or extend®Gold Antifade Reagent with DAPI (#8961).
- For best results, allow the mounting medium to set overnight at room temperature. Store slides flat at 4°C and protected from light for long-term storage.
Released November 2006
revised November 2013
Protocol identification: 24
Specificity/Sensitivity
The myosin IIa antibody detects endogenous levels of total myosin IIa protein. The antibody does not cross-react with non-muscle myosin IIb or IIc heavy chains.
Species Reactivity:
human, mouse, mouse
source / cleaning
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of mouse myosin IIa.
bottom
Non-muscle myosin is an actin-based motor protein essential for cell motility, cell division, migration, adhesion, and polarity. The holoenzyme consists of two identical heavy chains and two sets of light chains. Light chains (MLC) regulate myosin II activity and stability. Heavy chains (NMHC) are encoded by three genes,MYH9,MYH10, OfMYH14, which produce three different isoforms of non-muscle myosin II, IIa, IIb and IIc (reviewed in 1). Although the three isoforms perform the same enzymatic tasks, binding and contracting actin filaments that are coupled to ATP hydrolysis, their cellular functions do not appear to be redundant and they have distinct subcellular distributions (2-5). The carboxy-terminal tail domain of myosin II is important in isoform-specific subcellular localization (6). Research studies have shown that myosin IIa phosphorylation on Ser1943 helps regulate breast cancer cell migration (7).
- Conti, MA and Adelstein, R.S. (2008)cell science j121, 11-18.
- Sandquist, J.C. et al. (2006)JBiolChem281, 35873-83.
- Even-Ram, S. et al. (2007)natural cell biology9, 299-309.
- Vicente-Manzanares, M. et al. (2007)cell biology176, 573-80.
- Wylie, S.R. e Chantler, P.D. (2008)Mol Biol. Cell19, 3956-68.
- Sandquist, J.C. e Means, A.R. (2008)Mol Biol. Cell19, 5156-67.
- Dulyaninova, N.G. and others (2007)Mol Biol. Cell18, 3144-55.
signaling pathways and proteins
Discover pathways + proteins associated with this product.
upstream/downstream proteins
STRING - Known and predicted protein-protein interactions.
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FAQs
What is the function of myosin II? ›
Myosin II is implicated in the 'rounding up' of cells prior to cytokinesis, and is essential in the formation of the contractile cleavage furrow during cytokinesis. Now it appears that myosin II plays a role in all stages of cytokinesis, as a recent report suggests that myosin II drives post-mitotic cell spreading.
How is myosin II activated? ›Its activity is regulated on one hand by the Rho-activated serine–threonine kinase Rho-kinase which phosphorylates and inactivates myosin phosphatase, and on the other hand by the calcium-dependent enzyme myosin light chain kinase (MLCK) which phosphorylates MLC, resulting in myosin II activation.
What does non-muscle myosin II do? ›The actin motor protein non-muscle myosin II (NMII) acts as a master regulator of cell morphology, with a role in several essential cellular processes, including cell migration and post-synaptic dendritic spine plasticity in neurons.
Is myosin II a protein? ›Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties.
What is myosin IIA? ›Myosin IIA, a cytoplasmic myosin capable of forming bipolar filaments, is concentrated in the myoids of trout and bass cones, suggesting that muscle-like sliding filaments of actin and myosin might be responsible for cone contraction.
Where is myosin II found? ›Myosin II (also known as conventional myosin) is the myosin type responsible for producing muscle contraction in muscle cells in most animal cell types. It is also found in non-muscle cells in contractile bundles called stress fibers.
What is myosin II vs myosin V? ›Myosin V contains more light chains and a longer 'lever arm' relative to myosin II, which allows myosin V to move in larger steps along the actin filaments (reviewed in [7]). Myosin V may also colocalize with F-actin bundles.
What causes the power stroke for myosin II? ›The power stroke occurs when ADP and phosphate dissociate from the myosin head. The power stroke occurs when ADP and phosphate dissociate from the actin active site.
Is myosin II essential for cell migration? ›Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration.
How is non-muscle myosin II activated? ›Thus, non-muscle myosin II activation depends on RLC phosphorylation, which favours the myosin ATPase activity in the presence of actin.
What is the non-muscle myosin II gene called? ›
MYH9 is a large gene localized on chromosome 22q12. 3, spanning more than 106 kbp and composed of 41 exons. The open reading frame, spread from exon 2 to exon 41, encodes a protein of 1,960 amino acids, the non-muscle myosin heavy chain IIA (NMHC IIA) (Figure 1A).
What is myosin II made of? ›The myosin II molecule consists of two heavy chains and two pairs of light chains (called the essential and regulatory light chains). The heavy chains have globular head regions and long α-helical tails, which coil around each other (more...)
What does myosin II bind to? ›Myosin II minifilaments bind to F-actin via motor head domains, forming contractile structures important to cell shape change and motility of muscle and nonmuscle cells.
Where is myosin found in the body? ›Myosin is found in all cells in the human body but they are particularly abundant in muscle cells, where they are located in myofibrils, a major component of the contractile unit of a muscle cell. Myosin is a superfamily of proteins that play an important role in binding actin, hydrolyzing ATP and transducing force.
Is myosin II bipolar? ›On a molecular level, contraction derives from the bipolar structure of the myosin II filament in which the actin-binding motor domains are oriented at opposing filament ends.
What is myosin deficiency? ›Because of muscle weakness, affected individuals may start walking later than usual and have a waddling gait, trouble climbing stairs, and difficulty lifting the arms above shoulder level. Muscle weakness also causes some affected individuals to have trouble breathing.
What is the medical term for myosin? ›Medical Definition
myosin. noun. my·o·sin ˈmī-ə-sən. : actomyosin. : a fibrous globulin of muscle that can split ATP and that reacts with actin to form actomyosin.
Within the sarcomere, myosin slides along actin to contract the muscle fiber in a process that requires ATP. Scientists have also identified many of the molecules involved in regulating muscle contractions and motor behaviors, including calcium, troponin, and tropomyosin.
What are 4 functions of myosin? ›Myosins are involved in growth and tissue formation, metabolism, reproduction, communication, reshaping, and movement of all 100 trillion cells in the human body.
What is the Processivity of myosin II? ›Class II myosins are non-processive, exhibiting reduced gliding velocities at low motor density [5], [8] and single displacement events with optical trap measurements [102]. Two class I myosins, Myo1a and Myo1b, are also non-processive in single molecule assays [103].
Is myosin II only in animals? ›
Myosin II is also present in nonmuscle cells of animals and in organisms that predate animal evolution (e.g., fungi, amoebae), where, together with actin, it functions in cell division, cell migration, endocytosis and exocytosis, and changes in cell shape.
What is the difference between myosin 1 and 2? ›Myosin I is primarily involved in intracellular organization, but it also forms a critical component of small cell surface projections in intestinal cells. Myosin II can form higher order assemblies via the extended coiled-coil domains in the heavy chains.
What is the structure of myosin 2? ›Myosin II consists of pairs of heavy chains, essential light chains (ELCs) and regulatory light chains (RLCs), which combine to form the two-headed molecule and α-helical, coiled-coil tail (Fig. 1a). Each head comprises a motor domain and a regulatory domain, containing one RLC and one ELC.
Does myosin bind to troponin or tropomyosin? ›Tropomyosin blocks myosin binding sites on actin molecules, preventing cross-bridge formation, which prevents contraction in a muscle without nervous input. The protein complex troponin binds to tropomyosin, helping to position it on the actin molecule.
Does myosin bind to actin or troponin? ›Upon binding calcium, troponin moves tropomyosin away from the myosin-binding sites on actin (bottom), effectively unblocking it.
What are the domains of myosin II? ›Myosin has three main domains: a folded globular domain, the molecular motor that contains the ATPase and actin-binding properties, a lever arm, which binds a pair of light chains to amplify conformation changes in the motor domain, and a long α-helical tail that spontaneously forms a parallel, two-stranded, coiled- ...
What disease is myosin V? ›In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome.
How is non muscle myosin II activated? ›Thus, non-muscle myosin II activation depends on RLC phosphorylation, which favours the myosin ATPase activity in the presence of actin.
What protein is responsible for cell migration? ›Cells migrate along extracellular matrix (ECM) proteins using cell-surface receptors composed of integrins and proteoglycans. In various pathological conditions, such as cancer metastasis, cells restructure the ECM and reacquire the ability to migrate, but to detrimental effect.
What triggers cell migration? ›Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. Errors during this process have serious consequences, including intellectual disability, vascular disease, tumor formation and metastasis.