The electrophoretic profiles of the venom and RP-HPLC fractions were analyzed by SDS-PAGE under reducing conditions, following the protocol established by Laemmli (1970) [53 (link)]. Samples were loaded onto polyacrylamide gels with a stacking gel (5%) and resolving gel (12%) and subjected to electrophoresis under 100 V for 1 h. To estimate molecular mass, the ‘Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis’ (GE Healthcare, Chicago, IL, USA) was used. The gels were stained with Coomassie brilliant blue for visualization.
Amersham low molecular weight calibration kit for sds electrophoresis
Manufactured by GE Healthcare
Sourced in United States, Japan
The Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis is a tool used for molecular weight determination in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments. The kit contains a set of pre-stained protein markers with known molecular weights, which can be used as reference standards to estimate the molecular weights of unknown protein samples during electrophoresis.
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Lab products found in correlation
Gt f650, by Epson (2 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride pvdf membrane, by GE Healthcare (1 mentions)
Image lab analysis software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Quick cbb, by Fujifilm (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Knifetec 1095, by Foss (1 mentions)
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (1 mentions)
Xcell surelock mini cell, by Thermo Fisher Scientific (1 mentions)
Nupage 3 8 tris acetate protein gel, by Thermo Fisher Scientific (1 mentions)
Himark pre stained protein standard, by Thermo Fisher Scientific (1 mentions)
5 protocols using amersham low molecular weight calibration kit for sds electrophoresis
1
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Protocol
2
Profiling Acacia Pollen Allergens by SDS-PAGE
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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of Acacia pollen extract was performed using 12.5% acrylamide separation gels as described previously [8 (link)]. The protein bands from the electrophoresis of Acacia pollen extract or purified rAca f 1 were electro-transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, UK), and after blocking and washing, membranes were incubated with a serum pool or individual sera from patients with Acacia allergy or with control sera (1 : 5 dilutions) for three hours. Biotinylated goat anti-human IgE (Nordic-Mubio, Susteren, Netherlands) (1 : 1000 v/v in PBS) was added to detect specific IgE, and the blotted membrane strips were then incubated with 1 : 12,000 v/v in TPBS-HRP-linked streptavidin (Sigma-Aldrich, St. Louis, Mo, USA). Finally, strips were incubated with Super signal West Pico Chemiluminescent Substrate Kit (Thermo Scientific, Waltham, MA, USA) for five minutes, and proteins were then visualised by chemiluminescence using ChemiDoc XRS+ system (Bio-Rad). The molecular masses of protein bands were estimated using Image Lab Analysis Software (Bio-Rad) by comparison with protein markers of known molecular weights (Amersham Low molecular weight Calibration Kit for SDS electrophoresis, GE Healthcare, UK).
3
SDS-PAGE Analysis of Hb Derivatives
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All solutions of Hb derivatives were diluted with PBS to the Hb concentration of 0.012 mM. Then, each resulting solution was mixed with a half volume of the denaturing buffer 32 (0.19 M Tris-HCl buffer pH 6.8, containing 6.0% (wt/v) sodium dodecyl sulfate (SDS), 15% (v/v) 2mercaptoethanol, 30% (wt/v) sucrose, and 0.006% (wt/v) bromophenol blue) and incubated at 80 °C for 15 min. Electrophoresis was performed on a 13% polyacrylamide mini-slab gel using a mini-slab electrophoresis system (NA-1010; Nihon Eido Corp., Tokyo, Japan). An Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis (GE Healthcare Japan Ltd., Tokyo, Japan) was used for molecular weight markers. After the gels were stained with Coomassie brilliant blue (Quick CBB; Fujifilm Wako Pure Chemical Corp.), images of the stained gels were obtained using transmission scanning (GT-F650; Seiko Epson Corp., Nagano, Japan).
4
Quantification of Wheat Gluten Proteins
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Grain samples of 35 g from each cv. were milled to a fine powder with a Knifetec™ 1095 (Foss, Hillerød, Denmark). Gluten proteins (gliadins, HMW-GS, and LMW-GS) were extracted from 30-mg samples and quantified following a previous reported methodology [23] (link).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Mini-PROTEAN Tetra Cell (Bio-Rad) using 8% acrylamide gels for HMW-GS, and 12% acrylamide gels for gliadins and LMW-GS. Aliquots of 20 µg of each dried protein were suspended in 20 µL of loading buffer containing 20 g L -1 SDS, 0.2 g L -1 bromophenol blue, 1 mL L -1 β-mercaptoethanol, 0.05 mol L -1 Tris HCl (pH 6.8), and 100 mL L -1 glycerol, then boiled at 95 • C for 5 min before loading onto the gels. An Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis (MW 14,400-97,000 Da) (GE Healthcare, Chicago, IL, USA) was used to detect HMW, LMW-GS and gliadin bands. After electrophoretic separation at 40 mA, the gels were fixed in 70 mL L -1 acetic acid and 400 mL L -1 methanol and stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad). Image Lab 4.5.1 software (Bio-Rad) was used for relative quantification of the glutenin subunits on each gel. Gliadins were divided into three classes (ω, α/β and γ) based on their molecular weight.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Mini-PROTEAN Tetra Cell (Bio-Rad) using 8% acrylamide gels for HMW-GS, and 12% acrylamide gels for gliadins and LMW-GS. Aliquots of 20 µg of each dried protein were suspended in 20 µL of loading buffer containing 20 g L -1 SDS, 0.2 g L -1 bromophenol blue, 1 mL L -1 β-mercaptoethanol, 0.05 mol L -1 Tris HCl (pH 6.8), and 100 mL L -1 glycerol, then boiled at 95 • C for 5 min before loading onto the gels. An Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis (MW 14,400-97,000 Da) (GE Healthcare, Chicago, IL, USA) was used to detect HMW, LMW-GS and gliadin bands. After electrophoretic separation at 40 mA, the gels were fixed in 70 mL L -1 acetic acid and 400 mL L -1 methanol and stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad). Image Lab 4.5.1 software (Bio-Rad) was used for relative quantification of the glutenin subunits on each gel. Gliadins were divided into three classes (ω, α/β and γ) based on their molecular weight.
5
SDS-PAGE Analysis of Hemoglobin Derivatives
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All solutions of Hb derivative were diluted with PBS to the Hb concentration of 0.012 mM ([Hb] = 0.075 g/dL). Then, each resulting solution was mixed with a half volume of the denaturing buffer 31 (0.19 M Tris-HCl pH 6.8, 6.0% (wt/v) SDS, 15% (v/v) 2-mercaptoethanol, 30% (wt/v) sucrose, and 0.006% (wt/v) bromophenol blue) and was incubated at 80 °C for 15 min.
Electrophoresis was performed on a 13% polyacrylamide mini-slab gel using a mini-slab electrophoresis system (NA-1010; Nihon Eido Corp., Tokyo, Japan), or on a gradient gel (NuPAGE 3-8% Tris-Acetate Protein Gels; Invitrogen Corp., Thermo Fisher Scientific Inc., Waltham, U.S.A.) using a mini-slab electrophoresis system (XCell SureLock Mini-Cell; Invitrogen Corp., Thermo Fisher Scientific Inc.). An Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis (GE Healthcare Japan Ltd., Tokyo, Japan) and a HiMark Pre-Stained Protein Standard (Invitrogen Corp., Thermo Fisher Scientific Inc.) were used for molecular weight markers. After the gels were stained with Coomassie brilliant blue, images of the stained gels were obtained using transmission scanning (GT-F650; Seiko Epson Corp., Nagano, Japan).
Electrophoresis was performed on a 13% polyacrylamide mini-slab gel using a mini-slab electrophoresis system (NA-1010; Nihon Eido Corp., Tokyo, Japan), or on a gradient gel (NuPAGE 3-8% Tris-Acetate Protein Gels; Invitrogen Corp., Thermo Fisher Scientific Inc., Waltham, U.S.A.) using a mini-slab electrophoresis system (XCell SureLock Mini-Cell; Invitrogen Corp., Thermo Fisher Scientific Inc.). An Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis (GE Healthcare Japan Ltd., Tokyo, Japan) and a HiMark Pre-Stained Protein Standard (Invitrogen Corp., Thermo Fisher Scientific Inc.) were used for molecular weight markers. After the gels were stained with Coomassie brilliant blue, images of the stained gels were obtained using transmission scanning (GT-F650; Seiko Epson Corp., Nagano, Japan).
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