Labeling effects on the isoelectric point of green fluorescent protein

Richards, Dawn P.; Stathakis, Costas; Polakowski, Robert; Ahmadzadeh, Hossein; Dovic̀hi, Norman J.

doi:10.1016/s0021-9673(99)00687-1

Cited by 77 publications

(66 citation statements)

References 17 publications

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“…2 b and c). These have been reported in the literature (26,27). GFP was chosen in this example for its ability to be visualized by fluorescence during focusing.…”

Section: Resultsmentioning

confidence: 97%

Isoelectric focusing technology quantifies protein signaling in 25 cells

O'Neill¹,

Bhamidipati²,

Bi³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

187

234

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A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.cell signaling ͉ immunoassay ͉ phosphorylation ͉ Western blot ͉ microfluidic

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“…2 b and c). These have been reported in the literature (26,27). GFP was chosen in this example for its ability to be visualized by fluorescence during focusing.…”

Section: Resultsmentioning

confidence: 97%

Isoelectric focusing technology quantifies protein signaling in 25 cells

O'Neill¹,

Bhamidipati²,

Bi³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

187

234

View full text Add to dashboard Cite

show abstract

“…For a protein with m possible labeling sites, there are 2 m -1 possible fluorescent products [4]. These products can have dramatically different electrophoretic behavior, leading to an envelope of peaks producing complex electropherograms [4][5][6]. Most of our work has been with FQ, which converts cationic lysine residues into neutral products, which can be detected with exquisite sensitivity [7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Swearingen

Dickerson

Turner

et al. 2008

Journal of Chromatography A

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The fluorogenic reagent Chromeo P465 is considered for analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label α-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 × 10 −4 cm 2 V −1 s −1 . The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1 % with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM α-lactalbumin (r 2 = 0.99), with a 3σ detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, α-chymotrypsinogen A, and αlactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/ Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.

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“…Each of these products can have different electrophoretic or chromatographic properties, and labeled proteins can produce complex electropherograms during separation [26][27][28].…”

Section: Discussionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Wojcik

Swearingen

Dickerson

et al. 2008

Journal of Chromatography A

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Abstract3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), Chromeo P465, and Chromeo P503 are weakly fluorescent reagents that react with primary amines to produce fluorescent products. We studied the reaction of these reagents with α-lactalbumin by mass spectrometry. The reaction generated a set of products by the addition of one or more labels to the protein. At room temperature, the reaction was an order of magnitude faster with the Chromeo reagents than with FQ; however, the steady-state labeling efficiency was a factor of two higher for FQ compared with the Chromeo reagents. The relative abundance of the products with FQ usually followed a binomial distribution, which suggests that the labeling sites were uniformly accessible to this reagent. In contrast, the distribution of reaction products with the Chromeo reagents did not follow a binomial distribution for reactions performed in the absence of sodium dodecyl sulfate (SDS); it appears that the protein labeled with the Chromeo reagents refolded into a relatively stable secondary structure that hid some reactive sites. The reaction with the Chromeo reagent did follow the binomial distribution if the protein underwent treatment with 1% SDS at 95 °C for 5 min, which apparently disrupts the protein's secondary structure and allowed uniform access to all labeling sites. Chromeo 503 labeled seven of 17 the 13 primary amines in denatured α-lactalbumin.

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Labeling effects on the isoelectric point of green fluorescent protein

Cited by 77 publications

References 17 publications

Isoelectric focusing technology quantifies protein signaling in 25 cells

Isoelectric focusing technology quantifies protein signaling in 25 cells

Reaction of fluorogenic reagents with proteins

Reaction of fluorogenic reagents with proteins

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