For the analysis of specific enzymes partition in ATPS, the bottom phase of ATPS 2 (Table 2 ) was evaluated in SDS-PAGE electrophoresis. To prevent interference of salt and PEG in the method, the bottom phase had the phase formation exchanged by Milli-Q water using a 10.000 MWCO Amicon® Ultra Centrifugal Filters as described by the supplier. Retentate of this operation (protein enriched) was dissolved in Milli-Q water. Top phase could not be evaluated through this method because the 10.000 MWCO membrane was clotted with the high concentration of PEG 6000 in that phase. The samples were diluted times in NuPAGE™ LDS Sample Buffer (4X) (ThermoFisher Scientific, USA), and this mixture was heated at 70°C for 10 min. 12% (w/v) Bis-Tris polyacrylamide gel (NuPAGE™, 1.0 mm, 12-well) (ThermoFisher Scientific, USA) was loaded with 10 μL prepared samples and stained by GelCode™ Blue Safe Protein Stain (ThermoFisher Scientific, USA). Mark12™ Unstained Standard (ThermoFisher Scientific, USA) was used as molecular weight ladder consisting on the following sizes: 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6, 3.5, and 2.5 kDa. The protein concentration of the bottom phase sample preparation was 0.39 ± 0.02 mg/mL. The maximum protein load in the band was 0.5 μg.
Mark 12 unstained standard
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mark 12 Unstained Standard is a molecular weight marker used for protein size determination in SDS-PAGE. It consists of a mixture of 12 purified, unstained proteins with known molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Nupage novex bis tris 15 well precast gels, by Thermo Fisher Scientific (2 mentions)
Imagequant tl, by GE Healthcare (2 mentions)
Imagescanner 3, by GE Healthcare (2 mentions)
Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (1 mentions)
Pageruler plus, by Thermo Fisher Scientific (1 mentions)
Imagequant tl 7, by Cytiva (1 mentions)
Novex nupage gel system, by Thermo Fisher Scientific (1 mentions)
Nupage system, by Thermo Fisher Scientific (1 mentions)
Typhoon gel scanner, by Cytiva (1 mentions)
Nupage lds buffer, by Thermo Fisher Scientific (1 mentions)
Nupage novex, by Thermo Fisher Scientific (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
Nupage precast gel, by Thermo Fisher Scientific (1 mentions)
Pharosfx, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Sypro ruby protein stain, by Bio-Rad (1 mentions)
11 protocols using mark 12 unstained standard
1
SDS-PAGE Analysis of ATPS Partitioned Enzymes
2
SDS-PAGE Protein Separation and Analysis
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Protein eluates were mixed with SDS-PAGE loading buffer, heated for 5 min at 95 °C and loaded onto the 12% Tris-Glycine or 4–12% Bis-Tris polyacrylamide gel. Proteins were separated at 180 V until the dye front had reached the bottom of the gel. The molecular weight of proteins was estimated by running unstained (Mark12 Unstained Standard, Thermo Fisher Scientific) and pre-stained (PageRuler Plus, Thermo Fisher Scientific) marker proteins. Following electrophoresis, proteins were stained with either Coomassie Brilliant Blue, silver or subjected to Western blotting.
3
SDS-PAGE Analysis of Caspase-2 Variants
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Caspase-2 variants were detected on one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Stargardt et al. [44 (link)]. A ready-to-use molecular weight marker (Mark12™, Unstained Standard, Invitrogen, Waltham, MA, USA) was directly loaded as the size marker. The concentration of the soluble and insoluble fraction was calculated from densitometry analysis of SDS-PAGE gels using the software ImageQuantTL (7.0) (Cytiva, Uppsala, Sweden) via linear regression analysis. Purified T7AC-cpCasp2 (75, 50 and 25 µg/mL) was used as the standard. Two replicates of each sample were analyzed two times and the maximal coefficient of variation of 10% was used as acceptance criterion.
4
Protein Composition Analysis of Dried Aggregates
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The protein composition of the
freeze-dried aggregates, the acetone-dried aggregates, the hexane-dried
aggregates, as well as the protein composition in the supernatant
after centrifugation was analyzed under reducing conditions by SDS-PAGE
using the Novex NuPAGE gel system (Invitrogen, Thermo Fischer Scientific).
Samples were prepared by the addition of NuPAGE LDS sample buffer
(4×) and NuPAGE Reducing agent (10×) to a final protein
concentration of 2 mg/mL. Thereafter, samples were vortexed and heated
at 75 °C for 10 min in a water bath. After cooling, samples were
loaded into the wells of a NuPage 4–12% Bis-Tris gel. As a
running buffer, NuPAGE MES SDS (20×) was used, with antioxidant
in the cathode chamber. Electrophoresis was performed by applying
a constant voltage of 200 V for 40 min. Afterward, gels were stained
using coomassie blue (SimplyBlue). The apparent molecular weight of
the proteins present in each sample was determined by comparing the
position of the bands to a reference sample with proteins of various
molecular weights (Mark12 unstained standard, Invitrogen). The gels
were scanned in a densitometer (Gelscanner GS-900, Bio-Rad, Hercules,
CA, USA) with Image Lab software, which allows for the identification
of the proteins present in each sample.
freeze-dried aggregates, the acetone-dried aggregates, the hexane-dried
aggregates, as well as the protein composition in the supernatant
after centrifugation was analyzed under reducing conditions by SDS-PAGE
using the Novex NuPAGE gel system (Invitrogen, Thermo Fischer Scientific).
Samples were prepared by the addition of NuPAGE LDS sample buffer
(4×) and NuPAGE Reducing agent (10×) to a final protein
concentration of 2 mg/mL. Thereafter, samples were vortexed and heated
at 75 °C for 10 min in a water bath. After cooling, samples were
loaded into the wells of a NuPage 4–12% Bis-Tris gel. As a
running buffer, NuPAGE MES SDS (20×) was used, with antioxidant
in the cathode chamber. Electrophoresis was performed by applying
a constant voltage of 200 V for 40 min. Afterward, gels were stained
using coomassie blue (SimplyBlue). The apparent molecular weight of
the proteins present in each sample was determined by comparing the
position of the bands to a reference sample with proteins of various
molecular weights (Mark12 unstained standard, Invitrogen). The gels
were scanned in a densitometer (Gelscanner GS-900, Bio-Rad, Hercules,
CA, USA) with Image Lab software, which allows for the identification
of the proteins present in each sample.
5
SDS-PAGE Protein Separation Protocol
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Samples (55 μL) were prepared for SDS-PAGE by adding 20 μL NuPAGE (LDS) buffer (Thermo Scientific, Hertfordshire, UK), and 5 μL of either 0.5 M dithiothreitol (DTT) (reduced samples) or ultrapure water (non-reduced samples). Reduced samples were heated to 100 °C for 5 min and cooled to ambient temperature prior to loading to 4–12% Bis–Tris gels in a NuPAGE system (Invitrogen, Thermo-Fisher Scientific, Paisley, UK). Mark 12 Unstained Standard (Invitrogen) was used as molecular weight markers for protein-stained gels and SeeBlue pre-stained markers (Invitrogen, Loughborough, UK) were used for gels to be electroblotted. Proteins were separated according to the manufacturer’s instructions using 200 V, 350 mA and 100 W for 35 min. Gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid and after 1 h rinsed three times for 5 min each in deionised water before staining with Coomassie G-250 stain (SimplyBlue, Invitrogen). The gel was de-stained by rinsing with MilliQ water and imaged using a Typhoon gel Scanner (Amersham, UK).
6
Protein Separation by Gel Filtration
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Electrophoresis of sample was done and molecular weight was estimated by comparing sample protein bands with known molecular weight unstained ladder (Invitrogen Mark12 Unstained Standard) having 12 bands when run on gel in the range of 2.5 to 200 kDa. Isolation or separation of different molecular weight proteins was done by gel filtration chromatography (GFC). It separated proteins truly on the basis of molecular weight (Hagel 1998 ). Sephadex G-100 was ideal for 4 kDa to 150 kDa molecular weight protein and protein fractions to be separated were found to fall in this selected range.
7
Zein Protein Characterization by SDS-PAGE
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Zein doughs prepared as described above with water, hydrogen peroxide, peroxidase and hydrogen peroxide plus peroxidase were air-dried at ambient temperature. Additionally, zein with hydrogen peroxide addition was cast into films (plasticiser was not added) as described above.
SDS-PAGE was performed under non-reducing and reducing conditions using 4-12% polyacrylamide gradient gels (8 x 8 cm x1.0 mm thick with 15 wells) (NuPAGE® Novex, Invitrogen. Carlsbad, CA). Invitrogen Mark12 Unstained Standard was used. Samples were loaded to 10 g constant protein. Staining was with Coomassie Brilliant Blue R-250. After de-staining, the gels were photographed by scanning on a flatbed scanner.
SDS-PAGE was performed under non-reducing and reducing conditions using 4-12% polyacrylamide gradient gels (8 x 8 cm x1.0 mm thick with 15 wells) (NuPAGE® Novex, Invitrogen. Carlsbad, CA). Invitrogen Mark12 Unstained Standard was used. Samples were loaded to 10 g constant protein. Staining was with Coomassie Brilliant Blue R-250. After de-staining, the gels were photographed by scanning on a flatbed scanner.
8
SDS-PAGE Protein Characterization
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The protein preparations were characterized by SDS-PAGE under reducing and nonreducing conditions as described by A n y a n g o e t a l . ( 2 0 1 3 ) on pre-prepared 4-12% Bis-Tris (BT) gradient gels (Invitrogen Life Technologies, Carlsbad, CA) using an X Cell SureLock Mini-Cell electrophoresis unit (Invitrogen Life Technologies). The protein loading was ≈10 μg. Invitrogen Mark12 Unstained Standard was used. Protein bands were stained with Coomassie Brilliant Blue R-250 and photographed using a flat-bed scanner.
9
Quantitative Analysis of Protein Digestion
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The electrophoretic analyses were performed using 4-12% polyacrylamide NuPAGE® Novex® Bis-Tris 15 well precast gels (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. All samples were diluted 4-fold with NuPAGE® LDS sample buffer and then treated with 0.5 M DL-dithiothreitol and distilled water. Mark 12 Unstained Standard (Invitrogen) was used as a molecular weight (Mw) marker, as reference of the position of the bands. RGE and porcine pancreatin were deposed on the gels as controls, corresponding to the concentrations present in the digestive fluids. Gels were fixed in 30% (v/v) ethanol, 10% (v/v) acetic acid and 60% (v/v) deionized water and were rinsed for 15 min in deionized water before staining with Coomassie Blue. Image analysis of SDS-PAGE gels was carried out using Image scanner III (GE Healthcare Europe GbmH, Velizy-Villacoublay, France). After digitization of gels, the bands were selected and their gray intensity determined by densitometry using the software Image Quant TL™ (GE Healthcare Europe
GbmH, Velizy-Villacoublay, France). Densitometry analyses of the SDS-PAGE gels were used for semiquantification of protein levels. The percentage of each intact protein remaining in the gastric compartment at a given time was estimated in comparison with the undigested human milk.
GbmH, Velizy-Villacoublay, France). Densitometry analyses of the SDS-PAGE gels were used for semiquantification of protein levels. The percentage of each intact protein remaining in the gastric compartment at a given time was estimated in comparison with the undigested human milk.
10
SDS-PAGE Analysis of Meal and Digesta Proteins
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2.4.2.1. Protein analyses. SDS-PAGE was performed on meal and digesta samples using 4-12% polyacrylamide NuPAGE Novex bis-Tris 15-well precast gels (Invitrogen, Carlsbad, CA, USA). All samples were diluted with NuPAGE® LDS sample buffer and then treated with DL-dithiothreitol and deionized water. Ten µg of meal proteins was loaded into each well, thus taking into account the meal: secretion ratio in order to evaluate the sole impact of proteolysis. Mark 12 Unstained Standard (Invitrogen) was used as a molecular weight (Mw) marker. Gels were fixed in 30% (v/v) ethanol, 10% (v/v) acetic acid and 60% (v/v) deionized water and were rinsed in deionized water before staining with Bio-Safe Coomassie stain (Bio-Rad Laboratories, France). Discoloration of gels was performed with water. Image analyses of gels were carried out using Image scanner III (GE Healthcare Europe GbmH, Velizy-Villacoublay, France). Densitometry on bands was performed by measuring their grey intensity using the software Image Quant TL™ (GE Healthcare Europe183 GbmH, Velizy-Villacoublay, France). This allowed a semi-quantitative analysis of the digesta.
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