Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays

Calvert, Valerie; Tang, Yihui; Boveia, Vince; Wulfkuhle, Julie; Schutz-Geschwender, Amy; Olive, D. Michael; Liotta, Lance A.; Petricoin, Emanuel F.

doi:10.1385/cp:1:1:081

Cited by 36 publications

(23 citation statements)

References 33 publications

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“…Calf muscle homogenates were analyzed by the methods of Calvert et al (7). Homogenates were diluted in extraction buffer to 100 g protein/ml (final volume 250 -500 l) and then heated at 70°C for 2 h (Eppendorf Thermomixer 5436).…”

Section: Quantification Of Mnsod Hne Adducts and Carbonyl Groups Inmentioning

confidence: 99%

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Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

Pipinos

Swanson²,

Zhu³

et al. 2008

American Journal of Physiology-Regulatory, Integrative and Comp

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Section: Quantification Of Mnsod Hne Adducts and Carbonyl Groups Inmentioning

confidence: 99%

“…HNE adducts in homogenates of whole calf muscle from control (N ϭ 9) and ischemic (N ϭ 9) mice were measured by RPPA (7). HNE adducts expressed as HNE-BSA units/g protein were two times more abundant in homogenates of ischemic muscle (5.66 Ϯ 0.43) compared with control muscle (2.77 Ϯ 0.33).…”

Section: Ab Was Pretreated With 2 ϫ 10mentioning

confidence: 99%

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

Pipinos

Swanson²,

Zhu³

et al. 2008

American Journal of Physiology-Regulatory, Integrative and Comp

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“…Calvert et al reported that in their multiple dye experiment, background autofluorescence from the nitrocellulose and cellular material was dramatically lower at infrared wavelengths than at visible wavelengths. They reported that they could reduce background fluorescence of nitrocellulose at both 700nm and 800nm emission wavelengths in the near infrared spectra, when compared to visible wavelengths [35]. This suggests that, instead of the wavelength of 532nm for Qdot detection, we could use Qdots that emit at infrared wavelengths.…”

Section: Resultsmentioning

confidence: 99%

Quantum dots-based reverse phase protein microarray

Shingyoji

Gerion

Pinkel

et al. 2005

Talanta

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CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP) catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R 2 =0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.

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“…Near-infrared fluorescence allows the detection of attomol quantities of target molecules without signal amplification from protein patterns immobilized on a porous nitrocellulose support (21) with a sensitivity that is comparable to tyramide amplification and sufficient to assess the phosphorylation state in arrayed total proteins extracted from cancer biopsies (35). Unfortunately, the low proportion of target molecules in total protein lysates, used as solution analyte or as a printed analyte, strongly restricts the number of molecules that can be captured by the antibodies and thence be detected by near-infrared fluorescence.…”

Section: Discussionmentioning

confidence: 99%