A high-affinity reversible protein stain for Western blots

Antharavally, Babu S.; Carter, Brad; Bell, Peter A.; Mallia, A. Krishna

doi:10.1016/j.ab.2004.02.049

Cited by 58 publications

(24 citation statements)

References 21 publications

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“…An equal amount of protein of each sample was mixed with 6X Laemmli buffer, boiled for 5 min, and separated using SDS-PAGE (9% resolving gel) before being transferred to polyvinyl difluoride membranes. The MemCode protein stain was used to confirm equal loading (Antharavally et al 2004; Castorena et al 2014). Membranes were blocked in 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline, pH 7.5, plus 0.1% Tween-20) for 1 h at room temperature and transferred to 3% BSA-TBST with the appropriate primary antibody overnight at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…All data are shown as mean±SEM in the graph with representative blots above the bars and loading control below the bottom graph (n=10/group). MemCode, a protein stain used to confirm efficacy of transfer before the blocking step, was used as a loading control by quantitating the band at molecular weight 45 kD (Antharavally et al 2004; Castorena et al 2014). …”

Section: Figurementioning

confidence: 99%

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Prior treatment with the AMPK activator AICAR induces subsequently enhanced glucose uptake in isolated skeletal muscles from 24-month-old rats

Oki

Arias

Kanzaki

et al. 2018

Appl. Physiol. Nutr. Metab.

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5′ AMP-activated protein kinase (AMPK) activation may be part of the exercise-induced process that enhances insulin sensitivity. Independent of exercise, acute prior treatment of skeletal muscles isolated from young rats with a pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), causes subsequently improved insulin-stimulated glucose uptake (GU). However, efficacy of a single prior AICAR exposure on insulin-stimulated GU in muscles from old animals has not been studied. The purpose of this study was to determine whether brief, prior exposure to AICAR (3.5 hours before GU assessment) leads to subsequently increased GU in insulin-stimulated skeletal muscles from old rats. Epitrochlearis muscles from 24 month-old male rats were isolated and initially incubated ±AICAR (60 minutes), followed by incubation without AICAR (3 hours), then incubation ±insulin (50 minutes). Muscles were assessed for GU (via 3-O-methyl-[3H]-glucose accumulation) and site-specific phosphorylation of key proteins involved in enhanced GU, including AMPK, Akt, and Akt substrate of 160 kDa (AS160), via western blotting. Prior ex vivo AICAR treatment resulted in greater GU by insulin-stimulated muscles from 24 month-old rats. Prior AICAR treatment also resulted in greater phosphorylation of AMPK (T172) and AS160 (S588, T642 and S704). GLUT4 protein abundance was unaffected by prior AICAR and/or insulin treatment. These findings demonstrate that skeletal muscles from older rats are susceptible to enhanced insulin-stimulated GU after brief activation of AMPK by prior AICAR. Consistent with earlier research using muscles from young rodents, increased phosphorylation of AS160 is implicated in this effect that was not attributable to altered GLUT4 glucose transporter protein abundance.

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Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Prior treatment with the AMPK activator AICAR induces subsequently enhanced glucose uptake in isolated skeletal muscles from 24-month-old rats

Oki

Arias

Kanzaki

et al. 2018

Appl. Physiol. Nutr. Metab.

View full text Add to dashboard Cite

show abstract

“…The MemCode protein stain was used to confirm equal loading (36). Membranes were incubated with appropriate primary and secondary antibodies, and enhanced chemiluminescence of protein bands was quantified by densitometry (Alpha Innotech, San Leandro, CA) (34).…”

Section: Methodsmentioning

confidence: 99%

Postexercise Improvement in Insulin-Stimulated Glucose Uptake Occurs Concomitant With Greater AS160 Phosphorylation in Muscle From Normal and Insulin-Resistant Rats

et al. 2014

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Earlier research on rats with normal insulin sensitivity demonstrated that acute exercise increased insulin-stimulated glucose uptake (GU) concomitant with greater phosphorylation of Akt substrate of 160 kDa (pAS160). Because mechanisms for exercise effects on GU in insulin-resistant muscle are unknown, our primary objective was to assess insulin-stimulated GU, proximal insulin signaling (insulin receptor [IR] tyrosine phosphorylation, IR substrate 1–phosphatidylinositol-3-kinase, and Akt phosphorylation and activity), and pAS160 in muscles from acutely exercised (one session) and sedentary rats fed either chow (low-fat diet [LFD]; normal insulin sensitivity) or a high-fat diet (HFD; for 2 weeks, insulin-resistant). At 3 h postexercise (3hPEX), isolated epitrochlearis muscles were used for insulin-stimulated GU and insulin signaling measurements. Although exercise did not enhance proximal signaling in either group, insulin-stimulated GU at 3hPEX exceeded respective sedentary control subjects (Sedentary) in both diet groups. Furthermore, insulin-stimulated GU for LFD-3hPEX was greater than HFD-3hPEX values. For HFD-3hPEX muscles, pAS160 exceeded HFD-Sedentary, but in muscle from LFD-3hPEX rats, pAS160 was greater still than HFD-3hPEX values. These results implicated pAS160 as a potential determinant of the exercise-induced elevation in insulin-stimulated GU for each diet group and also revealed pAS160 as a possible mediator of greater postexercise GU of insulin-stimulated muscles from the insulin-sensitive versus insulin-resistant group.

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“…After treatment, protein removal from tooth surfaces was demonstrated by Ponceau S staining (16) and scanning electron microscopy (SEM). Ponceau S is a sodium salt of a diazo dye that is used as a stain for rapid reversible detection of protein bands on nitrocellulose or polyvinylidine fluoride membranes.…”

Section: Demonstration Of Protein Removal From Tooth Surfacementioning

confidence: 99%