Improved coomassie blue dye-based fast staining protocol for proteins separated by SDS-PAGE.

The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization

Comparison of the hemocompatibility of neurovascular flow diverters with anti-thrombogenic coatings

Despite the continual progress in technology, thromboembolic complications still occur following endovascular treatment of cerebral aneurysms and remain the primary contributor to morbidity and mortality. Thus, coating the surfaces of blood-contacting flow diverters may reduce thrombus-related complications in clinical use. In this study, the hemocompatibility of different commercially available flow diverters Pipeline Flex Embolization Device with Shield Technology (Medtronic), the p64 MW HPC (Phenox), and the DERIVO 2heal (Acandis) was investigated using an in vitro blood circulation model. The DERIVO 2 flow diverter was included in this study as an uncoated device. The markers of coagulation (thrombin-antithrombin complex (TAT)), thrombocytes (β-thromboglobulin (β-TG)), inflammation (PMN elastase), and complement system (SC5b-9, C3a, and C5a) activation were detected by enzyme-linked immunosorbent assay (ELISA). In addition to blood cell counts and hemolysis, thrombogenicity was determined using scanning electron microscopy. Flow diverters with anti-thrombogenic coatings resulted in significantly lower activation of coagulation (TAT) and platelets (β-TG) and lower adhesion of platelets compared with the uncoated DERIVO 2 flow diverter. There were no significant differences between the coated flow diverters in terms of TAT and β-TG levels, as well as platelet adhesion. However, significantly lower activation of the complement system (SC5b-9 and C5a) was observed for the DERIVO 2heal flow diverter in comparison with the Pipeline Flex Shield and the p64 MW HPC flow diverters emphasizing the improved hemocompatibility of the fibrin/heparin-coated DERIVO 2heal flow diverter

The influence of EDTA concentration on gel destaining.

<p>Illustrations of gel backgrounds after 1 hr incubation using different EDTA concentrations (0, 0.5, 1, 2, 4, 8, 16, 32, and 40 mM EDTA solutions, respectively) are shown (A). Average area intensity values and their standard deviations for gels destained with corresponding EDTA concentrations are presented as estimated using ImageJ software (B).</p

A comparison of bacterial (<i>E. coli</i>) proteins staining.

<p>Whole cell lysate (<i>E. coli</i>) was separated by 1D SDS-PAGE and gels were stained according to the original Dong protocol (lane 1) and according to our new proposed protocol with imidazole-zinc reverse staining followed by fast CBS and EDTA destaining (lane 2) to illustrate the effect of both protocols. Brightness and contrast of the magnified insets were adjusted for better illustrations.</p

2D SDS-PAGE of blood platelet, undepleted plasma, and rat brain tissue samples.

<p>The effect of an appropriate protocol on gel background destaining and spot detection is illustrated using blood platelet, undepleted plasma, and rat brain tissue samples analyzed by 2D SDS-PAGE. The gels were processed according to the original Dong (Protocol A) and the final (Protocol D) protocols. Magnified insets were exported from the Progenesis SameSpots software (brightness and contrast of the insets were adjusted by the software) and correspond to the gray areas highlighted in the gels. Spots that were detected in gels stained according to the final protocol D only are indicated with circles or arrows.</p

Staining of different protein fractions.

<p>1D SDS-PAGE gels were stained according to the original Dong protocol (lanes 1) and according to our new proposed protocol with imidazole-zinc reverse staining followed by fast CBS and EDTA destaining (lanes 2) to illustrate the effect of both protocols. To compare various protein types four different peripheral blood mononuclear cell protein fractions were used: cytosolic (A), nuclear (B), membrane (C), and cytoskeleton (D) protein fractions. Brightness and contrast of the magnified insets were adjusted for better illustrations.</p

The influence of temperature on gel destaining.

<p>Gels were destained with a boiling EDTA destaining solution for 1 hr (1); the combination of six washes in a boiling EDTA solution for 1 min followed by a 1 hr room temperature EDTA washing solution (2); a room temperature EDTA washing solution for 1 hr (3) or overnight (4); and the combination of six washes in a boiling EDTA solution for 1 min followed by a 6 hr room temperature washing (5). Illustrations of gel backgrounds after destaining with appropriate procedures are shown (A). Average area intensity values and their standard deviations for gels destained using appropriate procedures are presented as estimated using ImageJ software (B).</p

Comparing the effects of different protocols on the destaining of the gel background and detection of BSA bands.

<p>The effects of different protocols on gel background destaining was compared: gels were stained according to Dong <i>et al</i>. and destained six times for 1 min in boiling water, according to the original protocol (A); gels were stained according to Dong <i>et al</i>. and destained for 1 hr in boiling water (B); gels were stained according to Dong <i>et al</i>. and destained for 1 hr in a boiling EDTA solution (C); and gels were stained according to our new proposed protocol with imidazole-zinc reverse staining followed by fast CBS, and destained for 1 hr in a boiling EDTA solution (D). Gel background intensities were compared for all tested protocols (E), the results are expressed as mean values ± standard deviations from three independent experiments ; a, b, c, and d correspond to the above mentioned protocols A, B, C, and D, respectively. To estimate the differences in detection of BSA bands in gels stained according to the protocols A (blue), B (red), C (green), and D (purple), corresponding band areas were measured (F); the inset shows more detail illustration for lanes 5–8. The results are expressed as mean values ± standard deviations from three independent experiments. Two-fold serially diluted BSA samples were used for 1D SDS-PAGE and corresponded to 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 7.8 ng per lanes 1–8, respectively.</p

PLCL/PCL Dressings with Platelet Lysate and Growth Factors Embedded in Fibrin for Chronic Wound Regeneration

The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound

The Relation Between Protein Adsorption and Hemocompatibility of Antifouling Polymer Brushes

Whenever an artificial surface comes into contact with blood, proteins are rapidly adsorbed onto its surface. This phenomenon, termed fouling, is then followed by a series of undesired reactions involving activation of complement or the coagulation cascade and adhesion of leukocytes and platelets leading to thrombus formation. Thus, considerable efforts are directed towards the preparation of fouling-resistant surfaces with the best possible hemocompatibility. Herein, a comprehensive hemocompatibility study after heparinized blood contact with seven polymer brushes prepared by surface-initiated atom transfer radical polymerization is reported. The resistance to fouling is quantified and thrombus formation and deposition of blood cellular components on the coatings are analyzed. Moreover, identification of the remaining adsorbed proteins is performed via mass spectroscopy to elucidate their influence on the surface hemocompatibility. Compared with an unmodified glass surface, the grafting of polymer brushes minimizes the adhesion of platelets and leukocytes and prevents the thrombus formation. The fouling from undiluted blood plasma is reduced by up to 99%. Most of the identified proteins are connected with the initial events of foreign body reaction towards biomaterial (coagulation cascade proteins, complement component, and inflammatory proteins). In addition, several proteins that are not previously linked with blood-biomaterial interaction are presented and discussed