RFP-Booster_atto594

Application: Immunofluorescence (IF), Immunhistochemie (IHC)

Catalog no.: ABIN1082216

Additional Information

Description: Fluorescent fusion tags enable studying of protein localization and dynamics on cellular and molecular levels. Red fluorescent proteins (RFPs) are widely used as fluorescent tags. Commonly, a combination of red and green fusion proteins (GFPs) is employed to allow multicolor tracking and analysis of protein-protein interactions. However, the quantum yield and photostability of RFPs are often not sufficient for their efficient microscopic detection. This can be especially critical for the Super Resolution Microscopy applications (e.g. 3D-SIM or STED). Furthermore, many cell biological methods such as HCl treatment for BrdU-detection, the EdU-Click-iT™ treatment or heat denaturation for FISH lead to disruption of RFP signal.

Specificity: RFP-Booster efficiently highlights, enhancesand stabilizes monomeric red fluorescentproteins such as mRFP1, mCherry or mPlum but also mRuby

Comments:

Booster are very small, highly specific GFP- or RFP-binding proteins covalently coupled to the superior fluorescent dyes from ATTO-TEC.
With our Booster you reactivate, boost, stabilizate the signals of your fusion proteins.

Application Details

Assay Procedure: 1. Fixation: 4% paraformaldehyde (PFA) or 1:10 formalin (37% formaldehyde, 10-15% MetOH) in PBS, 10 min., RT.
2. Wash 3x with PBS containing 0.1% Tween 20 (PBST). Critical: do not let coverslips “dry”.
3. Permeabilisation: PBS containing 0.5% Triton X-100, 5 min., RT. Alternatively, permeabilise by incubating in 100% methanol for 5 min at -20°C.
4. Wash 2x with PBST.
5. Blocking: 4% BSA in PBST, 10 min, RT.
6. RFP-Booster incubation: dilute RFP-Booster 1:200 – 1:400 in blocking buffer and incubate 1 h, RT.
Note: For multiplexing protocols you can combine RFP-Booster with any other antibody.
7. Wash 3x 5-10 min in PBST.
8. If required, counterstain with DNA fluorescent dyes, e.g. DAPI.
9. Before mounting, coverslips can be very briefly rinsed in water to prevent salt crystals to form.
10. Mount in VectaShield (Vector Labs) or other mounting media with anti-fading agents and seal mounted coverslips with clear nail polish.

Application Notes: For the immunofluorescence staining of RFP-fusion proteins in fixed cells

Buffer: PBS

Preservative: 0.01% Sodium azide

Storage: Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze. Protect from light.

Restrictions: For Research Use only

Images

RFP-Booster specifically labels RFP-fusion proteins. HeLa cells expressing mRFP-PCNA, fixed and permeabilized. Specific staining of early, mid and late replication foci with RFP-Booster_Atto594 (dilution 1/200, 1 h at RT).

Enhancement of RFP signal with RFP-Booster_Atto594. Comparison of signal intensity of a HeLa cell line stably expressing a nuclear RFP-fusion protein before and after RFP-Booster treatment (1/200 dilution).

Improvement of RFP signal stability with RFP-Booster_Atto594. RFP fluorescence bleaches very quickly upon irradiation with high laser intensity. In contrast, fluorescent signal remains stable when enhanced with RFP-Booster_Atto594.

Publications

Product: Guizetti, Schermelleh, Mäntler et al.: "Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments." in: Science (New York, N.Y.), Vol. 331, Issue 6024, pp. 1616-20, 2011 (PubMed).

Cordes, Maiser, Steinhauer et al.: "Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy." in: Physical chemistry chemical physics : PCCP, Vol. 13, Issue 14, pp. 6699-709, 2011 (PubMed).

Ridzuan, Moon, Knuepfer et al.: "Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development." in: PLoS ONE, Vol. 7, Issue 3, pp. e33845, 2012 (PubMed).

Mikeladze-Dvali, von Tobel, Strnad et al.: "Analysis of centriole elimination during C. elegans oogenesis." in: Development (Cambridge, England), Vol. 139, Issue 9, pp. 1670-9, 2012 (PubMed).

Ries, Kaplan, Platonova et al.: "A simple, versatile method for GFP-based super-resolution microscopy via nanobodies." in: Nature methods, Vol. 9, Issue 6, pp. 582-4, 2012 (PubMed).