The samples of leaf were immediately harvested after a plant being uprooted, weighed and frozen in liquid nitrogen followed by grinding in chilled pestle and mortar to a fine powder. This powder was extracted with 40 mM (w/v) Tris-HCl, pH 7.5, 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was mixed with 6-X protein-dye containing 240 mM Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, 0.04% bromophenol blue and 5% beta-mercaptoethanol. The samples containing 40 μg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by Bradford method using BSA (bovine serum albumin) as a standard curve. After electrophoresis, the gels were stained with a commercial available silver stain according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
Silver stain
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Silver stain is a laboratory product used for the detection and visualization of proteins in polyacrylamide gels. It is a sensitive staining technique that utilizes silver ions to bind to and detect the presence of proteins in the gel.
Automatically generated - may contain errors
Lab products found in correlation
Protean 2, by Bio-Rad (2 mentions)
70.1 ti rotor, by Beckman Coulter (1 mentions)
Optima xe 90, by Beckman Coulter (1 mentions)
Pb221s, by Sartorius (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Black flat bottom high base 386 well plates, by Greiner (1 mentions)
Anykd, by Bio-Rad (1 mentions)
Pherastar plate reader, by BMG Labtech (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Ptch1, by Cell Signaling Technology (1 mentions)
Epcam, by Cell Signaling Technology (1 mentions)
Kif3b, by Cell Signaling Technology (1 mentions)
Flot1, by Cell Signaling Technology (1 mentions)
Icam 1, by Cell Signaling Technology (1 mentions)
Ift88, by Proteintech (1 mentions)
15 protocols using silver stain
1
Leaf Protein Extraction and SDS-PAGE
2
Purify Pf Extracellular Vesicles
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To purify
PfEVs, the frozen supernatant generated above was thawed on ice and loaded on quick-seal ultracentrifugation tubes (Bechman Coulter cat# 343322), then centrifuged at 150, 000g for 2hrs using an Optima XE90 ultracentrifuge and 70.1Ti rotor (Beckman Coulter). The pellet was washed twice by re-suspending in cold PBS and centrifuging at 150,000g for 2 hours after each wash. The final pellet was loaded onto OptiPrep™ density gradient medium prepared as described
47 (link)–
49 (link) and centrifuged at 250,000g for 18 hours. 1ml fractions were collected from the top of the gradient into 1.5ml Eppendorf tubes. To estimate the density of the purified vesicles, the weights of the tubes containing the fractions were measured using weighing machine (Sartorius, PB221S). Each fraction was diluted in PBS to 13.5ml and centrifuged at 150, 000g for 2 hours and the pellet re-suspended in 400µl of 8M urea, 2.5% SDS in 50 mM phosphate buffer, pH 8.0 to extract proteins, before concentrating using a 3KDa MWCO concentrator (Pierce™). A fifth of each fraction was analysed for the presence of proteins by running on SDS-PAGE and staining using silver stain (Bio-Rad).
PfEVs, the frozen supernatant generated above was thawed on ice and loaded on quick-seal ultracentrifugation tubes (Bechman Coulter cat# 343322), then centrifuged at 150, 000g for 2hrs using an Optima XE90 ultracentrifuge and 70.1Ti rotor (Beckman Coulter). The pellet was washed twice by re-suspending in cold PBS and centrifuging at 150,000g for 2 hours after each wash. The final pellet was loaded onto OptiPrep™ density gradient medium prepared as described
47 (link)–
49 (link) and centrifuged at 250,000g for 18 hours. 1ml fractions were collected from the top of the gradient into 1.5ml Eppendorf tubes. To estimate the density of the purified vesicles, the weights of the tubes containing the fractions were measured using weighing machine (Sartorius, PB221S). Each fraction was diluted in PBS to 13.5ml and centrifuged at 150, 000g for 2 hours and the pellet re-suspended in 400µl of 8M urea, 2.5% SDS in 50 mM phosphate buffer, pH 8.0 to extract proteins, before concentrating using a 3KDa MWCO concentrator (Pierce™). A fifth of each fraction was analysed for the presence of proteins by running on SDS-PAGE and staining using silver stain (Bio-Rad).
3
Quantification of Myosin Heavy Chain Isoforms
4
Protein Extraction from Rose Cultivars
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Protein extraction was done according to our previous methods [27 (link)]. The frozen powdered samples from all cut rose cultivars was extracted with extraction buffer (pH 7.5) containing 40 mM (w/v) Tris-HCl, pH 7.5, 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was mixed with protein-dye and 20 μg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by the Bradford method using BSA (bovine serum albumin) as a standard curve. After electrophoresis, the gels were stained with a commercial available silver stain according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
5
Proteinase K Sensitivity Assay
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For PK resistance assays, equal volumes of solubilized homogenates, Sarkospin supernatants or pellets fractions were treated or not with 1 µg.ml−1 Proteinase K (Sigma) from 0 to 60 min at 37 °C. At the end of the indicated time, samples were added Laemmli 1x prior to denaturation at 95 °C for 5 min, and loaded on Mini-Protean TGX 12% gels (Biorad) followed by SDS-PAGE electrophoresis. Gels were whether stained for total protein amount (silver stain, Biorad) or transferred on nitrocellulose 0.2 µm membranes with Trans-Blot Turbo transfer system (Biorad) using the Mixed molecular weight program. Membranes were fixed with PFA, and proteins were immunolabelled as described for filter trap.
6
Plant Protein Extraction and SDS-PAGE
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The frozen plant samples (leaf and root) were grinded in liquid nitrogen with pestle and mortar to a fine powder. The powdered samples were used for extraction of protein using protein extraction buffer containing 40 mM (w/v) Tris-HCl, pH 7.5, 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP (polyvinylpyrrolidone) and 1% (v/v) Triton X-100. The extracts was centrifuged at 13,000 rpm for 10 min at 4 °C and resulting supernatant was mixed with 2-X protein-dye containing 240 mM Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, 0.04% bromophenol blue and 5% beta-mercaptoethanol. The samples containing 10 μg of proteins quantified by Bradford [44 (link)] using BSA (bovine serum albumin) as a standard curve were loaded on 12.5% polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Subsequently, the gels were stained in commercial accessible silver stain according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
7
Kinetic Analysis of BRISC and BRCA1-A DUB Activity
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To assess chain length dependence of human BRISC and BRCA1-A (truncated RAP80) DUB activity, 4 μg K63-linked (Ub)2, (Ub)3, or (Ub)4 (Boston Biochem) were incubated with 5 nM DUB complex in 210 μl volume (gel filtration buffer). Samples (10 μl) were taken at regular intervals, reactions were stopped by addition of SDS-PAGE loading buffer and subsequently samples were analyzed qualitatively by SDS-PAGE (AnyKD, Bio-Rad) and silver stain (Bio-Rad). Initial velocity of cleavage of (Ub)2 at variable BRCA1-A concentrations (0-500 nM) was measured using 200 nM internally quenched K63-linked (Ub)2 as substrate in assay buffer (50 mM HEPES pH 7.0, 100 mM NaCl, 0.5% (w/v) BSA, 0.03% (w/v) Brij-35, 0.2 mM TCEP). Relative TAMRA fluorescence was monitored in 10 s time increments (excitation at 540 nm, emission at 590 nm) and assays were carried out at 22°C in black flat-bottom high-base 386-well plates (Greiner Bio-One) using a Pherastar plate reader (BMG Labtech). Initial velocity in relative fluorescence units per second was determined by linear regression.
8
Characterization of Bacterial Lipooligosaccharides
9
Diubiquitin Protease Assay Protocol
10
Proteomic Profiling of KIF3A/B-Deficient hPSCs
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