Stain-Free technology as a normalization tool in Western blot analysis

Guertler, Anne; Kunz, Nancy; Gomolka, Maria; Hornhardt, Sabine; Friedl, Anna A.; McDonald, K. W.; Kohn, Jonathan E.; Posch, Anton

doi:10.1016/j.ab.2012.10.010

Cited by 320 publications

(261 citation statements)

References 15 publications

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“…Prior to data analysis, critical protein load was determined to avoid saturated fluorescence signals by establishing a dose-dependent relationship between protein load and fluorescence signal. 27 Hepatic proteasome activity A total of 20 mg of frozen liver tissue was homogenized in 180 lL of ice-cold lysis buffer (pH 7.8) for 2 · 2 min at 25 Hz with the TissueLyser II and incubated on ice for 30 min. Subsequently, the homogenates were centrifuged (15,700 · g for 10 min at 4°C), and the supernatant was used.…”

Section: Hepatic Protein Expression Using Western Blottingmentioning

confidence: 99%

Restriction on an Energy-Dense Diet Improves Markers of Metabolic Health and Cellular Aging in Mice Through Decreasing Hepatic mTOR Activity

Schloesser

Campbell

Glüer

et al. 2015

Rejuvenation Research

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Dietary restriction (DR) on a normal low-fat diet improves metabolic health and may prolong life span. However, it is still uncertain whether restriction of an energy-dense, high-fat diet would also be beneficial and mitigate age-related processes. In the present study, we determined biomarkers of metabolic health, energy metabolism, and cellular aging in obesity-prone mice subjected to 30% DR on a high-fat diet for 6 months. Dietary-restricted mice had significantly lower body weights, less adipose tissue, lower energy expenditure, and altered substrate oxidation compared to their ad libitum-fed counterparts. Hepatic major urinary proteins (Mup) expression, which is linked to glucose and energy metabolism, and biomarkers of metabolic health, including insulin, glucose, cholesterol, and leptin/adiponectin ratio, were likewise reduced in high-fat, dietary-restricted mice. Hallmarks of cellular senescence such as Lamp2a and Hsc70 that mediate chaperone-mediated autophagy were induced and mechanistic target of rapamycin (mTOR) signaling mitigated upon high-fat DR. In contrast to DR applied in low-fat diets, anti-oxidant gene expression, proteasome activity, as well as 5¢-adenosine monophosphate-activated protein kinase (AMPK) activation were not changed, suggesting that high-fat DR may attenuate some processes associated with cellular aging without the induction of cellular stress response or energy deprivation.

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Section: Hepatic Protein Expression Using Western Blottingmentioning

confidence: 99%

Restriction on an Energy-Dense Diet Improves Markers of Metabolic Health and Cellular Aging in Mice Through Decreasing Hepatic mTOR Activity

Schloesser

Campbell

Glüer

et al. 2015

Rejuvenation Research

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show abstract

“…11972) purchased from Cell Signaling Technology. For all proteins, volume density of each target band was normalized to the total protein loaded into each lane using stain-free technology (18).…”

Section: Analytical Proceduresmentioning

confidence: 99%

Modulation of autophagy signaling with resistance exercise and protein ingestion following short-term energy deficit

Smiles

Areta

Coffey

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

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Autophagy contributes to remodeling of skeletal muscle and is sensitive to contractile activity and prevailing energy availability. We investigated changes in targeted genes and proteins with roles in autophagy following 5 days of energy balance (EB), energy deficit (ED), and resistance exercise (REX) after ED. Muscle biopsies from 15 subjects (8 males, 7 females) were taken at rest following 5 days of EB [45 kcal·kg fat free mass (FFM) Ϫ1 ·day Ϫ1 ] and 5 days of ED (30 kcal·kg FFM Ϫ1 ·day Ϫ1 ). After ED, subjects completed a bout of REX and consumed either placebo (PLA) or 30 g whey protein (PRO) immediately postexercise. Muscle biopsies were obtained at 1 and 4 h into recovery in each trial. Resting protein levels of autophagy-related gene protein 5 (Atg5) decreased after ED compared with EB (ϳ23%, P Ͻ 0.001) and remained below EB from 1 to 4 h postexercise in PLA (ϳ17%) and at 1 h in PRO (ϳ18%, P Ͻ 0.05). In addition, conjugated Atg5 (cAtg12) decreased below EB in PLA at 4 h (ϳ20, P Ͻ 0.05); however, its values were increased above this time point in PRO at 4 h alongside increases in FOXO1 above EB (ϳ22-26%, P Ͻ 0.05). Notably, these changes were subsequent to increases in unc-51-like kinase 1 Ser757 phosphorylation (ϳ60%) 1 h postexercise in PRO. No significant changes in gene expression of selected autophagy markers were found, but EGR-1 increased above ED and EB in PLA (ϳ417-864%) and PRO (ϳ1,417-2,731%) trials 1 h postexercise (P Ͻ 0.001). Postexercise protein availability, compared with placebo, can selectively promote autophagic responses to REX in ED. autophagy; skeletal muscle; resistance exercise; muscle protein breakdown MAINTENANCE AND REMODELING of skeletal muscle mass depends on the net balance between simultaneous processes of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Periods of energy deficit (ED) are associated with losses of lean body mass (35), and we (2) and others (44) have demonstrated that short-term ED through dietary restriction reduces postabsorptive rates of MPS compared with energy balance (EB). The protein degradation signaling pathways underlying this increased MPB response are not well characterized, although increased mRNA expression of ubiquitin proteasomal system (UPS) markers have been reported following short-term ED (9, 10). However, this UPS response is repressed following protein consumption (9), suggesting a possible muscle protein "sparing" effect of protein ingestion when in ED.The loss of muscle proteins resulting from ED (35) may, in part, be attributed to enhanced activity of the autophagylysosomal pathway (henceforth, termed autophagy). For example, chronic (6 mo) ED increases mRNA and protein markers of autophagy (53). Autophagy degrades, via lysosomal proteolysis, protein aggregates and damaged organelles that are then "recycled" to yield energy to maintain cellular metabolism (11). This process commences with the sequestration of a portion of cytoplasm by a double-membrane vacuole called the autophagosome, which subsequently releases its...

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“…Some researchers have stained the blots with total protein stains, such as Coomassie, Flamingo Pink, Sypro Ruby, Amido Black, Ponceau S, and stain-free technology, to measure the total protein signal in each lane as loading control 16,20,13,27,1,4,12 . The total protein loading control avoids the pitfalls associated with housekeeping proteins.…”

mentioning

confidence: 99%

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

Posch

Kohn

et al. 2013

JoVE

Self Cite

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The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.

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Stain-Free technology as a normalization tool in Western blot analysis

Cited by 320 publications

References 15 publications

Restriction on an Energy-Dense Diet Improves Markers of Metabolic Health and Cellular Aging in Mice Through Decreasing Hepatic mTOR Activity

Restriction on an Energy-Dense Diet Improves Markers of Metabolic Health and Cellular Aging in Mice Through Decreasing Hepatic mTOR Activity

Modulation of autophagy signaling with resistance exercise and protein ingestion following short-term energy deficit

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

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