Cell Cycle-Chromobody plasmid

Description
DNA plasmid encoding for anti-PCNA VHH (anti-PCNA Nanobody) fused to fluorescent protein TagRFP. PCNA is a cell cycle marker.

Specificity
PCNA (proliferating cell nuclear antigen), tested in human cells and zebrafish

Applications

Live cell imaging
Visualizing endogenous cell cycle marker protein PCNA
PCNA as biomarker

Cell Cycle-Chromobody plasmid
Cell Cycle-Chromobody plasmid (TagRFP)

Product Size Code Price Buy
Product Cell Cycle-Chromobody® plasmid (TagRFP) Size 20 µg Code ccr Price $ 999
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Cell Cycle-Chromobody format

 

Cell Cycle-Chromobody
plasmid (TagRFP)

Vector type

mammalian
expression vector

Promoter

constitutive CMV IE

Reporter

TagRFP

Codon usage

mammalian

Selection

Kan/Neo

Nuclear localization signal (NLS)

No

Please contact us for more details.

Specificity
Proliferating cell nuclear antigen (PCNA), tested in human cells & zebrafish

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

Sequence
With the Cell Cycle-Chromobody plasmid you receive the sequence information of the Alpaca antibody to PCNA fused to TagRFP, as well as the full vector sequence.

Microscopy techniques
Wide-field epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. STED and 3D-SIM; high-content microscopy and analysis

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

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

Burgess A, Lorca T, Castro A., Quantitative live imaging of endogenous DNA replication in mammalian cells. PLoS One. 2012;7(9):e45726. doi: 10.1371/journal.pone.0045726. Epub 2012 Sep 20. PMID: 23029203
Schorpp K, Rothenaigner I, Maier J, Traenkle B, Rothbauer U, Hadian K., A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells. J Biomol Screen. 2016 Oct;21(9):965-77. doi: 10.1177/1087057116641935. Epub 2016 Apr 4. PMID: 27044685

 

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 VHH 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.

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