Actin-Chromobody plasmids

Actin visualization in HeLa, PC3, U2OS, MEF, LNCaP, and BHK cells using Actin-Chromobody: Fluorescent microscopy images of cells transfected with Actin-Chromobody; green: TagGFP2, red: TagRFP (Evrogen), and blue: DAPI counterstain.

Nuclear Actin-Chromobody: NIH 3T3 cells (murine fibroblasts) transfected with the nuclear Actin-Chromobody (TagGFP2, green) and the Lamin-Chromobody (TagRFP, red) were starved in serum-free medium for 24 hours. Nuclear Actin-Chromobody probed endogenous F-actin assembly and distribution is shown 30 s after stimulation with serum. Courtesy of Matthias Plessner.

The time lapse analysis reveals the reorganization of actin after treatment with Cytochalasin D: HeLa cells were subjected to confocal imaging upon treatment with 2 µM of Cytochalasin D for 1 hour and recovery for 4 hours. Actin Chromobody enables monitoring of actin dynamics in real-time in living cells.

Description
DNA plasmid encoding for anti-Actin V_HH (anti-actin Nanobody) fused to a fluorescent protein.

Specificity
Actin microfilaments in human, rodent, plant cells, zebrafish, and Toxoplasma gondii

Applications
Live cell imaging
Visualizing actin dynamics
Actin as biomarker or control

Actin-Chromobody plasmids
Actin-Chromobody plasmid (TagGFP2)
Actin-Chromobody plasmid (TagRFP)
Nuclear Actin-Chromobody plasmid (TagGFP2)*

Product	Size	Code	Price	Buy
Product Actin-Chromobody® plasmid (TagRFP)	Size 20 µg	Code acr	Price $ 999	Buy +
Product Actin-Chromobody® plasmid (TagGFP2)	Size 20 µg	Code acg	Price $ 999	Buy +
Product Nuclear Actin-Chromobody® plasmid (TagGFP2)	Size 20 µg	Code acg-n	Price $ 999	Buy +

Actin-Chromobody formats

	Actin-Chromobody plasmid (TagGFP2)	Actin-Chromobody plasmid (tagRFP)	Nuclear Actin-Chromobody plasmid (TagGFP2)
Vector type	mammalian expression vector	mammalian expression vector	mammalian expression vector
Promoter	constitutive CMV IE	constitutive CMV IE	constitutive CMV IE
Reporter	TagGFP2	TagRFP	TagGFP2
Codon usage	mammalian	mammalian	mammalian
Selection	Kan/Neo	Kan/Neo	Kan/Neo
Nuclear localization signal (NLS)	No	No	Yes

Please contact us for more details.

Specificity
Actin microfilaments in human, rodent and plant cells, zebrafish, Toxoplasma gondii

Encoded Nanobody/ VHH
Monoclonal anti-Actin single domain antibody (sdAb) fragment

Sequence
With the Actin-Chromobody plasmid you obtain the sequence of the Alpaca antibody to Actin fused to TagRFP or TagGFP2, as well as the full vector sequence.

Microscopy techniques
Wide-field epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. STED

Transfection
Transfection of Chromobody plasmids into mammalian cells can be done with standard DNA-transfection methods, e.g. lipofection (Lipofectamine 2000® from Thermo Scientific), according to the manufacturer’s protocol for the transfection reagent. Please choose the transfection method that works the best for your cell type.

Storage instructions
Shipped at ambient temperature. Store at -20 °C

Brochure

Better images with Nanobodies (PDF)

Datasheet

SDS

EULA

Panza P, Maier J, Schmees C, Rothbauer U, Söllner C.
Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development. 2015 May 15;142(10):1879-84. doi: 10.1242/dev.118943. PMID: 25968318

Rocchetti A, Hawes C, Kriechbaumer V.
Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody. Plant Methods. 2014 May 19;10:12. doi: 10.1186/1746-4811-10-12. eCollection 2014. PMID: 24872838

Periz J, Whitelaw J, Harding C, Gras S, Del Rosario Minina MI, Latorre-Barragan F, Lemgruber L, Reimer MA, Insall R, Heaslip A, Meissner M.
Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. Elife. 2017 Mar 21;6. pii: e24119. doi: 0.7554/eLife.24119. PMID: 28322189

Johann G. Danzl, Sven C. Sidenstein, Carola Gregor, Nicolai T. Urban, Peter Ilgen, Stefan Jakobs & Stefan W. Hell
Coordinate-targeted fluorescence nanoscopy with multiple off states. Nature Photonics 10, 122–128 (2016) doi:10.1038/nphoton.2015.266

Melak M, Plessner M, Grosse R.
Actin visualization at a glance. J Cell Sci. 2017 Feb 1;130(3):525-530. doi: 10.1242/jcs.189068. Epub 2017 Jan 12. Review. PMID: 28082420

Plessner M, Melak M, Chinchilla P, Baarlink C, Grosse R.
Nuclear F-actin formation and reorganization upon cell spreading. J Biol Chem. 2015 May 1;290(18):11209-16. doi: 10.1074/jbc.M114.627166. Epub 2015 Mar 10. PMID: 25759381

Baarlink C, Plessner M, Sherrard A, Morita K, Misu S, Virant D, Kleinschnitz EM, Harniman R, Alibhai D, Baumeister S, Miyamoto K, Endesfelder U, Kaidi A, Grosse R.
A transient pool of nuclear F-actin at mitotic exit controls chromatin organization. Nat Cell Biol. 2017 Nov 13. doi: 10.1038/ncb3641. [Epub ahead of print] PMID:29131140

Are Chromobodies constitutively expressed?

Yes, Chromobody expression is regulated by immediate early promotor CMV. This promotor allows constitutive Chromobody expression.

Do Chromobodies only work in live cells?

Yes, the Chromobody plasmid is only expressed in live cells. Cells should be transfected with the Chromobody plasmid at least overnight to observe the Chromobody location signal. Alternatively, cells can be fixed prior to imaging.
Note: We don't recommend fixation of cells for the Histone-Chromobody.

When should I image my cells after transfection with the Chromobody plasmid?

The Chromobody signal is maintained up to 3 days in the cell. However, this also depends strongly on the cell type.
We recommend to image the cells 16-24 hours after transfection.

Do the Chromobodies diffuse through the cell membrane into growth medium?

No, Chromobodies are small proteins being expressed in the cytosol. They are not secreted into the medium and remain in the cell as long as the cell maintains its plasma membrane integrity.

Are Chromobodies fluorogenic or do they only emit fluorescence when bound to a target?

Chromobodies are chimeric proteins consisting of a V_HH fused to a fluorescent protein. They maintain their fluorescence regardless of whether they are bound to a target or not.

Can I amplify the Chromobody plasmid in bacteria?

Yes, the Chromobody plasmids can be propagated in E.coli by standard techniques.

*The nuclear Actin-Chromobody^® plasmid has been published and described by Prof. Dr. Robert Grosse, Institute of Pharmacology, BPC, University of Marburg, Germany under license from ChromoTek:

Matthias Plessner, Michael Melak, Pilar Chinchilla, Christian Baarlink, Robert Grosse (2015). Nuclear F-actin formation and reorganization upon cell spreading J Biol Chem. (PubMed)

Only for research applications, not for diagnostic or therapeutic use!