Elsevier

Analytical Biochemistry

Volume 276, Issue 2, 15 December 1999, Pages 129-143
Analytical Biochemistry

Regular Article
A Luminescent Ruthenium Complex for Ultrasensitive Detection of Proteins Immobilized on Membrane Supports

https://doi.org/10.1006/abio.1999.4364Get rights and content

Abstract

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium–aluminum–garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25–1 ng protein/mm2) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

References (41)

Cited by (143)

  • Multiple scanning electrochemical microscopy mapping of tyrosinase in micro-contact printed fruit samples on polyvinylidene fluoride membrane

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    Citation Excerpt :

    Among the different employed membranes for protein blotting and detection, polyvinylidene fluoride (PVDF) is widely used since it is a highly hydrophobic and porous support with a superior protein binding capacity and a remarkable mechanical and chemical stability. Moreover, PVDF maintains the enzymatic activity of adsorbed proteins and is compatible with different protein labeling protocols including the ones based on Coomassie Blue, silver or gold staining and fluorescent or chemiluminescent dyes [10–16]. Recently, proteins immobilized on PVDF membranes have also been detected and employed for the visualization of human finger prints by scanning electrochemical microscopy (SECM) [17].

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To whom correspondence should be addressed at Bioanalytical Assay Development Group, Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402. Fax: 541-344-6504. E-mail: [email protected].

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