SDS-PAGE analysis was conducted on the various digesta samples as well as the initial undigested matrices, after dilution of all samples to 0.5% protein in milliQ water and then further diluted 1:2 in 0.1 mol/L Na-phosphate buffer, pH 7.0. The samples were then mixed 1:1 with 2 × Laemmli sample buffer (20 μL) containing DTT (350 mmol/L) and placed on a heat block (80 °C, 4 min). Protein standards (10 μL, Precision Plus Protein™ Dual Xtra #1610377, BIO-RAD) and the test samples (20 μL) were loaded onto a NuPAGE™ 4–12% Bis-Tris gel (10 well, 1.0 mm, Invitrogen™). Dithiothreitol 99% (DTT, 457779, Sigma-Aldrich), 2x Laemmli Sample Buffer (BIO-RAD, #1610737), NuPAGE™ 4–12% Bis-Tris gel 10 well 1.0 mm (Invitrogen™), NuPAGE™ MOPS SDS Running Buffer (20x, Invitrogen™), Precision Plus Protein™ Dual Xtra (BIO-RAD, #1610377), SimplyBlue™ SafeStain (Invitrogen™). The electrophoresis (40 min, 200V, 120 mA [240 mA for 2 gels], 25W) was performed using NuPAGE™ MES SDS running buffer. The gel was rinsed two times in water, followed by staining with Invitrogen™ SimplyBlue™ SafeStain (60 min, 22 °C). The gel was de-stained in water (100 mL, 1 hour). A NaCl solution (20% w/v, 20 mL) was added to the de-staining water and the gel was incubated overnight. Gel Imaging were performed on a Gel Doc™ EZ System (BIO-RAD) using Image Lab™ software.
Precision plus protein dual xtra
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
The Precision Plus Protein™ Dual Xtra is a pre-stained protein standard used for molecular weight determination and monitoring protein separation in SDS-PAGE and Western blotting applications. It contains a mixture of ten recombinant proteins with molecular weights ranging from 2 to 250 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Image lab 6, by Bio-Rad (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
Trupage lds sample buffer, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Chemidoc mp instrument, by Bio-Rad (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
7 protocols using precision plus protein dual xtra
1
SDS-PAGE Analysis of Protein Digests
2
SDS-PAGE Gel Electrophoresis Protocol
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The SDS-PAGE gel electrophoresis (15%) was performed according to [57 (link),58 (link)]. A Biorad Precision Plus Protein Dual Xtra (2–250 kDa) molecular weight standard was used as a marker. Gels were stained with Coomassie R-250 and analyzed using Bio-Rad’s Image Lab 6.1 software (Bio-Rad Laboratories, Inc., Berkeley, CA, USA, 2020).
3
Identification of Bacteriolytic Proteins via SDS-PAGE
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SDS PAGE was used for the identification of higher molecular weight antimicrobial proteins (bacteriolysins). Cultures were grown overnight in broth and the cell free supernatants were prepared as described above. The proteins from the bacterial supernatant were precipitated by the addition of ammonium sulphate salts up to a concentration of 50%. The precipitate was collected by centrifugation and resuspended in water. Supernatants were then incubated with TruPAGETM LDS sample buffer (Sigma-Aldrich, Wicklow, Ireland) for 10 minutes at 70°C. Samples were run on 12% acrylamide gels at 30 mA, together with Precision Plus Protein™ Dual Xtra prestained protein standards (Bio-Rad, Hertfordshire, UK) which were used to estimate molecular mass with a range of 2–250 kDa. The completed gels were divided in two, one half was stained using the EZBlueTM staining reagent (Sigma-Aldrich). The other half was washed with 1% tween-80 (Sigma-Aldrich) for 45 minutes, followed by three 5 minute washes in distilled water. This gel was overlaid with soft MRS agar (0.8% agar), seeded with 0.25% of an overnight culture of L. delbrueckii subsp. bulgaricus LMG 6901. The plate was incubated overnight to determine the mass of any antimicrobial proteins produced.
4
Western Blot Quantification Protocol
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For western blot, a standard SDS-PAGE gel was prepared according to the Laemmli protocol20 (link) with modifications according to Ladner et al.21 (link). Precision Plus Protein™ Dual Xtra (Bio-Rad Laboratories) was used as molecular weight standard in 1:10 dilution. The proteins in the gel were transferred to a low-fluorescence PVDF blotting membrane in a semi-dry blot procedure (15 min, 25 V. 1.3 A, Trans-Blot® TurboTM instrument, Bio-Rad Laboratories) using the Trans-Blot® TurboTM RTA transfer kit. The membrane was blocked with AdvanBlock™ -PF blocking solution for 30 min, incubated with “8-4-4” anti-His-tag antibody (3.3 mg/L in blocking solution, 60 min) and washed with AdvanWashTM washing solution (3 × 5 min). Then, the membrane was incubated with secondary RPE-goat-anti-mouse-IgG (1:4000 in washing buffer with 0.5% milk powder, 60 min) and washed with washing solution (2 × 5 min) and finally with Tris buffer (20 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, 1 × 5 min). Fluorescence detection was performed using the Chemi-Doc MP instrument (Bio-Rad Laboratories) by measuring the Cy3 channel (605/50 nm, recording time 5.33 s).
5
Protein Profiling of Venom
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Protein patterns from all venoms were observed in 12% SDS-PAGE according to Sambrook [109 ] and stained with Coomassie colloidal stain [84 (link)]. We used Image Lab 6.0.1 (BioRad, Hercules, CA, USA) to process apparent molecular weight for the bands in SDS-PAGE and zymography using 5 µL Precision Plus Protein™ Dual Xtra (BioRad, Hercules, CA, USA) as the molecular weight marker.
6
Cashew Protein Extraction and Characterization
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Protein extract was prepared from fresh milled raw cashew nuts as described by Wangorsch et al.25 and its concentration was determined by Bradford according to manufacturer's instructions. SDS‐PAGE protein separation was carried out on NuPAGE 1 mm 10% Bis‐Tris gels (Novex by Life Technologies) under non‐reducing conditions by loading 10–100 μg of denatured cashew protein in NuPAGE LDS sample buffer alongside a Precision Plus Protein Dual Xtra molecular weight marker (Bio‐Rad Laboratories Inc., CA). Gels were either stained with Bio‐Safe™ Coomassie Stain (Bio‐Rad Laboratories Inc.) or subjected to western blotting as previously described.62 Blotting was carried out using specific Bet v 1 (BETVIA, rabbit polyclonal antibody, orb51330; dilution 1:1,000; Biorbyt, Cambridge, United Kingdom) and Ara h 8 (rabbit polyclonal antibody, PA‐AH8, dilution 1:1,000; Indoor Biotechnologies, Cardiff, United Kingdom) antibodies alongside 10 μg of a native Bet v 1 and recombinant Ara h 8 positive control (NA‐BV1‐1 and RP‐AH8, respectively; Indoor Biotechnologies). Imaging and analysis were performed using a Universal Hood III and Image Lab 4.1. software (Bio‐Rad Laboratories Inc.).
7
Purification and Characterization of GasE
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During the purification process, the RPC-FPLC eluted fractions of GasE were analyzed in duplicate by Tris-Tricine SDS-PAGE, using an 18 % acrylamide resolving gel [27 (link)]. After electrophoresis at 100 mV for 2 h, one gel was silver stained while the other was used to detect the inhibitory activity in an overlay assay as described previously [24 (link)]. L. paracasei C1351 was used as the indicator strain. The Precision Plus Protein Dual Xtra (Bio-Rad) was used as molecular weight standards.
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