Visible fluorescent detection of proteins in polyacrylamide gels without staining

Ladner, Carol L.; Yang, Jing; Turner, Raymond J.; Edwards, Robert A.

doi:10.1016/j.ab.2003.10.047

Cited by 248 publications

(220 citation statements)

References 20 publications

(34 reference statements)

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“…Samples were diluted in 100 nmol/L potassium acetate #bib3 mmol/L magnesium acetate, 20 mmol/L Tris (pH7.4) and 100 μ g of total protein was run on a 12% polyacrylamide gel. In‐gel protein loading and sample transfer was checked with 2 #bib2 #bib2‐Trichloroethanol as described 29. Membranes were blocked with 5% nonfat‐dry‐milk and incubated with the primary antibody (Table S3).…”

Section: Methodsmentioning

confidence: 99%

Megalencephalic leukoencephalopathy with cysts: the Glialcam‐null mouse model

Bugiani

Dubey

Breur

et al. 2017

Ann Clin Transl Neurol

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ObjectiveMegalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile‐onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion‐water homeostasis is only partly defined. We characterized Glialcam‐null mice for further studies.MethodsWe investigated the consequences of loss of GlialCAM in Glialcam‐null mice and compared GlialCAM developmental expression in mice and men.Results Glialcam‐null mice had early‐onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam‐null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC‐2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1‐interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years.Interpretation Glialcam‐null mice replicate the early stages of the human disease with early‐onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC‐2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

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Section: Methodsmentioning

confidence: 99%

Megalencephalic leukoencephalopathy with cysts: the Glialcam‐null mouse model

Bugiani

Dubey

Breur

et al. 2017

Ann Clin Transl Neurol

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“…The LLD for low MW protein standards (Pharmacia) was 200 ng (Trp content 0.8-2.3%) and 20 ng protein (4.5% Trp). The LDR was between 0.2 and 2 μg protein/band (R=0.99) and between 0.7 and 100 ng of Trp mass/protein [195]. It was undeniable that sensitive protein detection was highly dependent on the Trp content of proteins and hence resulted in a high degree of inter-protein variability.…”

Section: Additional Fluorescent Stainsmentioning

confidence: 96%

“…This procedure for in-gel protein detection is, however, very lengthy and requires up to two weeks for completion. Yet with the addition of 2,2,2-trichloroethanol to the gel matrix, native fluorescence protein detection could be carried out immediately after electrophoresis at 300 nm [195]. The LLD for low MW protein standards (Pharmacia) was 200 ng (Trp content 0.8-2.3%) and 20 ng protein (4.5% Trp).…”

Section: Additional Fluorescent Stainsmentioning

confidence: 99%

“…Not only was a filter (Corning 051) required to remove excitation energy but since the Gilford was designed to read absorbance, it was necessary to calculate the antilogarithm of peaks to obtain a measurement of signal. It gradually became more common to transilluminate gels with UV light to detect fluorescently stained proteins [20,157,158,182,184,186,190,191,195,246,247]. Subsequently, photographs of transilluminated gels had to be scanned by a densitometer before quantitative analysis could be undertaken; again, all without the aid of local computers that are now considered standard lab equipment.…”

Section: Equipment Innovationsmentioning

confidence: 99%

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Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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“…Muscle lysates were diluted 1:3 with 100% glycerol/Laemmli sample buffer (50/50) and run on 8% selfcast stain-free gels containing 0.5% 2,2,2-trichloroethanol (36). A total of 3 mg of lysate protein was separated for ;16 h at 140 V as previously described (37).…”

Section: Mhc Determinationmentioning

confidence: 99%

Human Muscle Fiber Type–Specific Insulin Signaling: Impact of Obesity and Type 2 Diabetes

et al. 2014

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Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulinmediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber type-specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemiceuglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1a (PDH-E1a) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1a). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1a) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.Skeletal muscle is important for whole-body insulinstimulated glucose disposal (1), and skeletal muscle insulin resistance is a common phenotype of obesity and type 2 diabetes (T2D) (2). Skeletal muscle is a heterogeneous tissue composed of different fiber types, which can be divided according to myosin heavy chain (MHC) isoform expression. Studies in rodents show that insulin-stimulated glucose uptake in the oxidative type I fiber-dominant muscles is higher than in muscles with a high degree of glycolytic type II fibers (3-6). Whether this phenomenon is due to differences in locomotor activity of individual muscles or a direct consequence of the fiber-type composition is largely unknown. In incubated rat muscle, insulin-induced glucose uptake was higher (;100%) in type IIa (oxidative/glycolytic) compared with IIx and IIb (glycolytic) fibers (7,8), suggesting that insulin-mediated glucose uptake is related to the oxidative capacity of the muscle fiber. In humans, a positive correlation between proportions of type I fibers in muscle and whole-body insulin sensitivity has been demonstrated (9-11). Furthermore, insulin-stimulated glucose transport in human muscle strips was associated with the relative type I fiber content (12). Thus, it is likely that human type I fibers are more important than type II fibers for maintaining glucose homeostasis in response to insulin. Indeed, a decreased proportion of type I fibers has been found in various insulin resistant states such as the metabolic syndrome (9), obesity (13,14), T2D in some (10,13,14) but not all (12,15) studies and following bedrest (16), as well as in tetraplegic patients (17), ...

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Visible fluorescent detection of proteins in polyacrylamide gels without staining

Cited by 248 publications

References 20 publications

Megalencephalic leukoencephalopathy with cysts: the Glialcam‐null mouse model

Megalencephalic leukoencephalopathy with cysts: the Glialcam‐null mouse model

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Human Muscle Fiber Type–Specific Insulin Signaling: Impact of Obesity and Type 2 Diabetes

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