The two-dimensional gel electrophoresis was performed with Ready prepTM2D starter kit (Bio-Rad). Antigen samples were purified by micro-bio-spin chromatography column (Bio-Rad) to remove salts. 40 μg of protein were run on 7 cm IPG strip (pH 3–10, 7 cm, Bio-rad) according to protocol mentioned in ready prep TM2D starter kit in PROTEIN IEF cell in step wise fashion viz. step1: 250V, linear for 20 minutes; step 2: 4,000V, linear for 150 minutes; step 3: VH-10000, rapid; step 4: Hold Step-500V for 4 hrs. The IPG strip was subjected to SDS-PAGE (2nd Dimension). 12% resolving gel was prepared and poured in the Bio-Rad gel cast. IPG strip was laid onto the polyacrylamide gel bed. It was electrophoresed in Tris-glycine SDS-PAGE running buffer (1X) at constant 200V for 40 minutes. Silver staining of the electrophoresed gel was performed using Plus One Silver Staining Kit (GE healthcare) following manufacturer’s manual. MS compatible staining procedure for gel was adapted from Plus One Staining Kit manual (Amersham). By omitting the glutaraldehyde from the sensitizer and formaldehyde from the silver solution, the method becomes compatible for mass spectrometry analysis, however, at the expense of sensitivity (10 ng). Therefore, only the prominent spots were carefully excised from the gel.
Plusone silver staining kit
Manufactured by GE Healthcare
Sourced in Sweden, United States
The PlusOne Silver Staining Kit is a laboratory equipment product designed for the detection of proteins in gel electrophoresis. The kit provides a set of reagents and solutions to perform silver staining, a sensitive method for visualizing proteins separated by SDS-PAGE or other electrophoretic techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Micro bio spin chromatography column, by Bio-Rad (1 mentions)
Ipg strip, by Bio-Rad (1 mentions)
Ready preptm2d starter kit, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
L 90k ultracentrifuge, by Beckman Coulter (1 mentions)
Enhanced chemiluminescence western blotting detection reagent, by GE Healthcare (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Phastgel, by GE Healthcare (1 mentions)
Native buffer strips, by GE Healthcare (1 mentions)
Plusone, by GE Healthcare (1 mentions)
Ampholine pagplate, by GE Healthcare (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Easy nanolc 2, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos etd, by Thermo Fisher Scientific (1 mentions)
24 protocols using plusone silver staining kit
1
2D-Gel Electrophoresis for Protein Analysis
2
SDS-PAGE Protein Separation and Detection
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Extracted proteins were supplemented with 5x-SDS sample buffer (10% SDS, 20% Glycerol, 100 mM Tris pH 7, 0.1% bromophenol blue; 25% 2-mercaptoethanol) and denatured for 10 min at 95 °C. Subsequently, the proteins were separated by SDS-PAGE43 (link). The polyacrylamide gels were stained for 10 min in FastGene® Q-Stain while shaking and afterward rinsed two times with H2O. PlusOne Silver Staining Kit (Ge Healthcare) was used to detect low abundant proteins. The resulting signals were recorded with the ImageQuant LAS 4000 and quantified with the ImageQuant TL (GE Healthcare) or the Fiji (http://fiji.sc ) image processing applications.
3
Comparative Proteomic Analysis of Porcine Muscle Quality
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High quality longissimus dorsi muscles (HQLD) and low quality longissimus dorsi muscles (LQLD) tissues were collected from Duroc pigs. Total protein isolation was performed using PRO-PREP protein extraction solution (iNtRON biotechnology, Sungnam, Korea) according to the manufacturer’s instructions. Concentrations of eluted proteins were measured using Pierce BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). Equal amounts of protein samples were precipitated with cold acetone. Protein pellets dissolved in 1× sodium dodecyl sulfate (SDS) sample buffer were separated by 8% and 12% SDS-polyacrylamide gel electrophoresis (PAGE). Following SDS-PAGE, protein spots were visualized using protocols described in PlusOne Silver staining kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). The complete protocol was followed to analyze gels. To prepare gels, the protocol was modified so that glutaraldehyde was omitted from the sensitization step and formaldehyde was omitted from the silver reaction step (Yan et al., 2000 (link)). Silver-stained gels were scanned (UMAX PowerLook 2100KL Imaging system, UMAX, Taiwan) and protein profiles were compared.
4
Sucrose Gradient Fractionation of PMP22 Liposomes
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After dialysis, a 750-μl sample of 20-μg PMP22 reconstituted into liposomes and dialysis buffer containing 50% (w/v) sucrose was prepared. This sample was placed at the bottom of a 5-ml polypropylene ultracentrifuge tube. A 750-μl sample of 20-μg PMP22 in DM micelles and dialysis buffer containing 50% sucrose was placed in another ultracentrifuge tube as a control. Dialysis buffer (3.38 ml) containing 40% sucrose was layered on top of the liposome or micelle layer, followed by 750 μl of dialysis buffer containing no sucrose. This created a stepwise sucrose gradient. All layers in the micelle sample contained 0.05% DM so as to not dilute the DM below the critical micelle concentration. Samples were then centrifuged at 160,000g at 4°C for 16 hours using a Beckman L-90K Ultracentrifuge equipped with an SW 55 Ti rotor. After ultracentrifugation, samples were collected from the sucrose gradient in 500-μl fractions from the top down using a glass Hamilton syringe; the fractions were analyzed by SDS-PAGE, followed by silver staining (GE Healthcare PlusOne Silver Staining Kit).
5
Western Blotting and Silver Gel Staining
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Western blotting was performed as previously described.86 (link) Briefly, boiled samples (10 min, 100°C) were loaded onto a 4%–12% Bis-Tris NuPAGE Novex gel (Invitrogen), followed by transfer onto a 0.45-μm membrane using the Novex system from Life Technologies (XCell II blot module). The membrane was then incubated with blocking solution for 1 h at RT before incubation with the appropriate primary Ab overnight at 4°C. The membrane was then incubated for 1 h with the appropriate secondary Ab (horseradish peroxidase [HRP]-conjugated Ab, 1:50,000). The signal was visualized using enhanced chemiluminescence western blotting detection reagents (GE Healthcare). For silver gel staining, the same procedure was followed without the transfer onto a membrane. The gel was fixed overnight after migration in a mixture containing 40% ethanol and 10% acetic acid. Proteins were revealed by silver staining using the PlusOne silver staining kit and following the manufacturer’s procedures (GE Healthcare).
6
Native Gel Electrophoresis of Core-Antibody Complexes
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Samples were mixed to achieve a final concentration of 10 µM coreS (as trimer, unless stated otherwise) and appropriate molar ratio of antibody as indicated. This concentration was chosen because it represents a good detection limit to differentiate between the binding of neutralizing and non-neutralizing Fabs and ScFvs. They were incubated at room temperature for 30 min and 2 µl of sample was layered for electrophoresis on a 20% homogeneous Phastgel using 1 µl/eight-lane applicators (unless stated otherwise) and native buffer strips (GE Healthcare). Gels were stained in Phastgel Blue R, destained and digitized. For detection at lower concentrations, 4 µl/six-lane applicators and PlusOne silver staining kit (GE Healthcare) were used.
7
Bovine Adipose Protein Profiling
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Adipose tissues were collected from cows, steers, and bulls. Total protein isolation was performed using PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. Proteins eluted were measured using Pierce BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). Equal amounts of protein samples were precipitated with cold acetone. Protein pellets were dissolved in 1× sodium dodecyl sulphate (SDS) sample buffer and separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, protein spots were visualized using protocols described in the PlusOne Silver staining kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). Complete protocol was followed for analytical gels. For preparative gels, the protocol was modified. Glutaraldehyde was omitted from the sensitization step. Formaldehyde was omitted from the silver reaction step (Yan et al., 2000 (link)). Silver-stained gels were scanned (UMAX PowerLook 2100KL Imaging system, UMAX, Taiwan) and protein profiles were compared.
8
Native-PAGE Electrophoresis and Enzyme Activity Staining
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Native-PAGE electrophoresis was performed using a 12.5% polyacrylamide gel at pH 8.8, with the Laemmli buffer system without SDS [41 (link)]. The protein was silver-stained using the PlusOne silver staining kit (GE Healthcare, Uppsala, Sweden). Enzyme activity staining was performed according to the method described by Hassan et al. [39 (link)] with slight modifications. The activity staining of the gel was performed in the presence of 1.0 mM of trans, trans-farnesol in 100 mM of glycine-NaOH buffer (pH 9.5) containing 54 μM of 1-methoxy phenazine methosulphate, 0.3 mM of nitroblue tetrazolium, and 1.0 mM of NAD+ at 35°C for 2 h.
9
Visualizing Anticandidal Bacteriocin Proteins
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The selected anticandidal bacteriocins were analyzed using glycine or tricine sodium dodecyl sulfate-polyacrylamide gel (Mini-Protein™ IV, BIO-RAD laboratory, Hercule, CA, USA) with a multicolor low-range protein ladder (Spectra™, Fermentus, Vilnius, Lithuania). They were visualized using the PlusOne Silver Staining Kit (PlusOne™, GE HEALTHCARE (AB)”, Pollards Wood, Nightingales Lane, Chalfont St. Giles, Buckinghamshire, HP8 4SP, UK). The molecular weights of the protein bands were calculated.
10
Protein Identification by Silver Staining
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Protein samples after SDS-PAGE were stained using the PlusOne™ silver staining kit (GE Healthcare). The stained protein bands of interest were identified by 1D-LC-ESI-Ion Trap-mass spectrometry analysis in the Proteomic Laboratory for System Biology Research in Hong Kong Baptist University.
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