Myosin IIa antibodies (2023)

support data

PM (kDa)230

Application Key:

  • WB-Western spot
  • IP- Immunoprecipitation
  • IHC- Immunhistochemie
  • Switch- Immunoprecipitation of chromatin
  • Database-Just-Point
  • 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

transmitted west1:1000
Immunofluorescence (immunocytochemistry)1:50

Save on computer

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.



Ver >Reduce >

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 page for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your western blot are now included in one convenient kit:#12957Solution kit for western blot applications

USE: Prepare solutions with deionized reverse osmosis water (RODI) or equivalent water.

  1. 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.
  2. 10X Tris-buffered saline (TBS): (#12498) To prepare 1L of 1X TBS: add 100mL of 10X to 900mL of dH2Hallo Mix.
  3. 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
  4. 10x Tris-Glycin SDS Laufpuffer: (#4050) To prepare 1L of 1X Run Buffer: Add 100mL of 10X Run Buffer to 900mL of dH2Hallo Mix.
  5. 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.
  6. 10X Tris-buffered saline with interpolation®20 (TBST): (#9997) Preparation of 1L 1X TBST: Add 100mL 10X TBST to 900mL dH2Hallo Mix.
  7. Magermilchpulver: (#9999).
  8. 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.
  9. wash buffer: (#9997) 1X TBS.
  10. Rinderserum albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; For 20mL add 1.0g BSA to 20mL 1X TBST and mix well.
  12. Biotinylated protein ladder detection package: (#7727).
  13. Prestained blue protein marker, large range (11-250 kDa): (#59329).
  14. membrane and blotting paper: (#12369) This protocol has been optimized for nitrocellulose membranes. In general, a pore size of 0.2 µm is recommended.
  15. HRP conjugated secondary antibody: anti-rabbit IgG, antibody linked to HRP (#7074).
  16. detection reagent: Reaktives SignalFire™ ECL (#6883).

B. Proteintransfer

A general protocol for sample preparation.

  1. Treat the cells by adding fresh media with buffer for the desired time.
  2. Aspirate the culture medium; wash cells with 1X PBS; striving.
  3. 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
  4. Sonicate for 10-15 seconds to complete cell lysis and DNA cleavage (to reduce sample stickiness).
  5. Heat a 20 L sample at 95–100 °C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. 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.

  8. 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

  1. (Optional) After transfer, wash the nitrocellulose membrane with 25 mL of TBS for 5 min at room temperature.
  2. Incubate the membrane in 25 mL of blocking buffer for 1 hour at room temperature.
  3. Wash three times for 5 min each with 15 mL of TBST.

II. Primary Antibody Incubation

  1. 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.
  2. Wash three times for 5 min each with 15 mL of TBST.
  3. 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.
  4. Wash three times for 5 min each with 15 mL of TBST.
  5. Proceed with screening (Section D).

D. Proteinnachweis

Instructions for use:

  1. Wash the membrane-bound HRP (conjugated antibody) three times for 5 min in TBST.
  2. 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.
  3. 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-ID: 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 with deionized reverse osmosis water (RODI) or equivalent water.

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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
  6. 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.

  1. 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.

  2. Allow cells to sit at room temperature for 15 min.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. 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.

  1. Block the sample in blocking buffer for 60 min.
  2. During the block, prepare the primary antibody by diluting it as directed on the product page under Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate the sample in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1 to 2 hours at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslips with prolong®Gold-Anti-Fade-Reagenz (#9071) or extend®Gold Antifade Reagent with DAPI (#8961).
  9. 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


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.


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 all three isoforms perform the same enzymatic tasks, tying and contracting actin filaments coupled with ATP hydrolysis, their cellular functions do not appear to be redundant and 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).

  1. Conti, MA and Adelstein, R.S. (2008)cell science j121, 11-18.
  2. Sandquist, J.C. et al. (2006)JBiolChem281, 35873-83.
  3. Even-Ram, S. et al. (2007)natural cell biology9, 299-309.
  4. Vicente-Manzanares, M. et al. (2007)cell biology176, 573-80.
  5. Wylie, S.R. e Chantler, P.D. (2008)Mol Biol. Cell19, 3956-68.
  6. Sandquist, J.C. e Means, A.R. (2008)Mol Biol. Cell19, 5156-67.
  7. 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

Myosin IIa antibodies (1)

STRING - Known and predicted protein-protein interactions.

database links

UniProt Identification:P35579


Veja with PhosphoSitePlus®

limited uses

Unless expressly agreed in writing and signed by a legally authorized representative of CST, the following terms apply to products supplied by CST, its affiliates or its distributors. Terms additional to or different from Customer's terms and conditions contained herein shall be rejected and shall not apply unless accepted in writing by a legally authorized representative of CST.

Products are labeled for research use only or with a similar labeling statement and have not been approved, approved, or cleared for any purpose by the FDA or any other foreign or domestic regulatory agency. The customer will not use any product for diagnostic or therapeutic purposes or in any other way that is contrary to the labeling. Products sold or licensed by CST are made available to Customer as an end user and solely for research and development purposes. Any use of the product for diagnostic, prophylactic, or therapeutic purposes, or any purchase of the product for resale (alone or as a component) or for any other commercial purpose requires a separate license from CST. Customer will not (a) sell, license, loan, donate, or transfer or make available to any third party any Products, alone or in combination with other materials, or use the Products to create commercial products, (b) copy, modify, develop Reverse, decompile, disassemble, or attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing products or services that may compete with CST's products or services, (c) the Products not alter or remove therefrom any trademark, trade name, logo, notice or patent or copyright mark; (d) use the Products only in accordance withCST Product Terms of Saleand comply with all applicable documentation and (e) any licenses, terms of use or similar agreements relating to any third party products or services used by Customer in connection with the Products.

For research use only. Do not use in diagnostic procedures.

Cell Signaling Technology is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. visit ourBrand Informationbook page.

Top Articles
Latest Posts
Article information

Author: Msgr. Refugio Daniel

Last Updated: 04/14/2023

Views: 5567

Rating: 4.3 / 5 (74 voted)

Reviews: 89% of readers found this page helpful

Author information

Name: Msgr. Refugio Daniel

Birthday: 1999-09-15

Address: 8416 Beatty Center, Derekfort, VA 72092-0500

Phone: +6838967160603

Job: Mining Executive

Hobby: Woodworking, Knitting, Fishing, Coffee roasting, Kayaking, Horseback riding, Kite flying

Introduction: My name is Msgr. Refugio Daniel, I am a fine, precious, encouraging, calm, glamorous, vivacious, friendly person who loves writing and wants to share my knowledge and understanding with you.