High-efficiency staining of proteins on different blot membranes

Yonan, Christopher R.; Duong, Phu; Chang, F. N.

doi:10.1016/j.ab.2004.11.010

Cited by 28 publications

(12 citation statements)

References 6 publications

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“…The same control extract was loaded in all gels to assure comparability between gels . Proteins were transferred to Immun‐Blot PVDF Membranes and subsequently stained with Reactive Brown 10 to control for differences in loading and transfer efficiency, and the bands captured with the ChemiDocTM Touch Imaging System and quantified with the Image Lab 5.2.1 software. The membranes were blocked for 1 hour in 4% bovine serum albumin in Tris‐buffered saline containing 0.1% Tween‐20 (TBS‐T; BSA‐blocking buffer) and incubated overnight at 4°C with the anti‐sarcolipin antibody (catalog no.…”

Section: Methodsmentioning

confidence: 99%

Sarcolipin expression in human skeletal muscle: Influence of energy balance and exercise

Morales-Álamo

Martinez‐Canton²,

Gelabert‐Rebato

et al. 2019

Scandinavian Med Sci Sports

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Sarcolipin (SLN) is a SERCA uncoupling protein associated with exercise performance and lower adiposity in mice. To determine SLN protein expression in human skeletal muscle and its relationship with adiposity, resting energy expenditure (REE), and performance, SLN was assessed by Western blot in 199 biopsies from two previous studies. In one study, 15 overweight volunteers underwent a pretest followed by 4 days of caloric restriction and exercise (45‐minute one‐arm cranking + 8‐hour walking), and 3 days on a control diet. Muscle biopsies were obtained from the trained and non‐exercised deltoid, and vastus lateralis (VL). In another study, 16 men performed seven sessions of 4‐6 × 30‐sec all‐out sprints on the cycle ergometer with both limbs, and their VL and triceps brachii biopsied pre‐ and post‐training. SLN expression was twofold and 44% higher in the VL than in the deltoids and triceps brachii, respectively. SLN was associated with neither adiposity nor REE, and was not altered by a severe energy deficit (5500 kcal/day). SLN and cortisol changes after the energy deficit were correlated (r = .38, P = .039). SLN was not altered by low‐intensity exercise in the overweight subjects, whereas it was reduced after sprint training in the other group. The changes in SLN with sprint training were inversely associated with the changes in gross efficiency (r = −.59, P = .016). No association was observed between aerobic or anaerobic performance and SLN expression. In conclusion, sarcolipin appears to play no role in regulating the fat mass of men. Sprint training reduces sarcolipin expression, which may improve muscle efficiency.

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Section: Methodsmentioning

confidence: 99%

Sarcolipin expression in human skeletal muscle: Influence of energy balance and exercise

Morales-Álamo

Martinez‐Canton²,

Gelabert‐Rebato

et al. 2019

Scandinavian Med Sci Sports

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“…Initial studies concerned the 100KF material that contained hemopexin and impurities as described above, and subsequent studies involved purified hemopexin preparations that SDS-PAGE analysis showed to contain various amounts of protein contaminants when stained with Ponceau S, 44 a protein stain that is less sensitive than Coomassie Blue. 81 A protocol for further fractionation of the 100KF fraction 44 used Blue-Sepharose chromatography, 11 a procedure shown to produce hemopexin contaminated with hyaluronidase, 73 to obtain samples containing hemopexin as well as multiple protein bands of >85 kDa. An alternative approach was also taken to fractionate a human 100KF preparation further by chromatography on a rabbit anti-hemopexin antibody affinity column, but the resulting product exhibited substantial cleavage following this treatment [Ref.…”

Section: Hemopexin As a Serine Proteasementioning

confidence: 99%

An alternative view of the proposed alternative activities of hemopexin

2011

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Hemopexin is a plasma protein that plays a well-established biological role in sequestering heme that is released into the plasma from hemoglobin and myoglobin as the result of intravascular or extravascular hemolysis as well as from skeletal muscle trauma or neuromuscular disease. In recent years, a variety of additional biological activities have been attributed to hemopexin, for example, hyaluronidase activity, serine protease activity, proinflammatory and anti-inflammatory activity as well as suppression of lymphocyte necrosis, inhibition of cellular adhesion, and binding of divalent metal ions. This review examines the challenges involved in the purification of hemopexin from plasma and in the recombinant expression of hemopexin and evaluates the questions that these challenges and the characteristics of hemopexin raise concerning the validity of many of the new activities proposed for this protein. As well, an homology model of the three-dimensional structure of human hemopexin is used to reveal that the protein lacks the catalytic triad that is characteristic of many serine proteases but that hemopexin possesses two highly exposed Arg-Gly-Glu sequences that may promote interaction with cell surfaces.

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“…Amido black is another alternative staining protocol to the Ponceau S staining method and is also a recommended method for staining blots [ 3 ]. Amido Black Staining Solution is designed for rapid staining of protein bands on nitrocellulose membranes.…”

Section: Introductionmentioning

confidence: 99%

Protein Stains to Detect Antigen on Membranes

D’Souza

Scofield

2015

Methods in Molecular Biology

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Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

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High-efficiency staining of proteins on different blot membranes

Cited by 28 publications

References 6 publications

Sarcolipin expression in human skeletal muscle: Influence of energy balance and exercise

Sarcolipin expression in human skeletal muscle: Influence of energy balance and exercise

An alternative view of the proposed alternative activities of hemopexin

Protein Stains to Detect Antigen on Membranes

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