Native polyacrylamide gel electrophoresis was conducted in an Invitrogen XCell SureLock using NativePAGE 3 to 12% bis-tris mini protein gels with 1X NativePAGE Anode Buffer and 1X NativePAGE Cathode Buffer. 5.2 μg of each sample was mixed with 4X NativePAGE sample buffer and then loaded onto the gel. Native PAGE was carried out at a constant voltage of 150 V for 1 hour followed by an additional 1 hour run at 250 V at 4°C. NativeMark Unstained Protein Standard was used as a protein ladder. After the run, gel was soaked in water to remove dye, and then stained with Coomassie Blue for protein detection.
Xcell surelock
Manufactured by Thermo Fisher Scientific
Sourced in United States
The XCell SureLock is a laboratory instrument designed for electrophoresis. It is capable of running sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tetra cell, by Bio-Rad (2 mentions)
Benzonase, by Merck Group (2 mentions)
Digitonin, by Merck Group (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nupage reducing agent 10, by Thermo Fisher Scientific (1 mentions)
Mab1501, by Merck Group (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Ab28481, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by AppliChem (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
T5168, by Merck Group (1 mentions)
27 g needle, by Terumo (1 mentions)
Ecl plus western blotting detection kit, by Cytiva (1 mentions)
Ab173838, by Abcam (1 mentions)
Bcatm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Procise protein sequencing system 492a, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
23 protocols using xcell surelock
1
Native Polyacrylamide Gel Electrophoresis
2
SDS-PAGE Analysis of MC-BMP2
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The obtained MC-BMP2 was solubilized in 50 μL of distilled water (DW). One microliter of NuPAGE LDS buffer (4×) (Invitrogen; CA, USA) and 2.5 μL of NuPAGE reducing agent (10×) (Invitrogen; CA, USA) were added to 6.5 μL of MC-BMP2 solution. For the control, 1 μL of NuPAGE LDS buffer and 2.5 μL of NuPAGE with 500 mM dithiothreitol as reducing agent were added to 2 μL of native BMP2-OpgY (1 μg/μL) solution. Native BMP2-OpgY and MC-BMP2 were individually incubated at 70 °C for 15 min and then loaded onto a NuPAGE 4–12% Bis-Tris gel (Invitrogen; CA, USA), followed by electrophoresis at 200 mV using XCell SureLock (Invitrogen; CA, USA). MC-BMP2 and native BMP2 were visualized by staining with 0.025% Coomassie Blue.
3
Liver Protein Expression Analysis
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Total liver and isolated primary hepatocyte lysates were prepared using radioimmunoprecipitation assay buffer with a protease-inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein (30 μg) was placed in the wells of 4–12% Bis-Tris Gels (Invitrogen, Carlsbad, CA, USA) and resolved. The products were then blotted onto a polyvinylidene difluoride membrane using NuPage transfer buffer and XCell SureLock (both from Invitrogen) and incubated with the following specific antibodies at 4 °C overnight: FAS (1:1000; #3180; Cell Signaling Technology, Danvers, MA, USA), SREBP-1 (1:1000; AB28481; Abcam, Cambridge, UK), and β-actin (1:3000; MAB1501; Millipore, Billerica, MA, USA).
4
Quantification and Visualization of LEDGF Hybrids
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Protein concentration of 1% SDS (AppliChem, Leuven, Belgium) protein extracts sheared with a 27 G needle (Terumo, Leuven, Belgium) was determined using a bicinchoninic acid (BCA) protein assay (Pierce, Aalst, Belgium). Proteins were separated on a 12.5% w/v SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (PVDF; BioRad) using an XCell SureLock electrophoresis system (Invitrogen). LEDGF-hybrids were detected using 1/2.000 polyclonal rabbit anti-LEDGF480–530 antibody (A300-848a; Bethyl Laboratories-Imtec Diagnostics N.V., Antwerpen, Belgium) and 1/5 000 secondary antibody (polyclonal goat anti-rabbit antibody coupled with horse radish peroxidase (HRP); Dako). Chemiluminescence was measured using a ECL plus western blotting detection kit (Amersham Biosciences, Roosendaal, The Netherlands). Equal loading was verified with a primary monoclonal antibody directed to α-tubulin (mouse, 1/10 000, 1 h at room temperature; T5168, Sigma-Aldrich) and secondary antibody in blocking buffer (1/10 000, polyclonal goat-anti mouse labelled with HRP; Dako). Visualization was done by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo scientific).
5
Neurexin-1 Expression in GPCs
6
Protein Sequencing of PVY and VLP
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PVY and VLP samples were analyzed under reducing conditions with SDS-PAGE using NuPAGE 4 to 12% bis-tris protein gel (Invitrogen). Wet electroblotting was carried out to transfer proteins from the gel to the polyvinylidene difluoride membrane. The transfer sandwich (filter paper–gel–membrane–filter paper) was placed into the transfer box (XCell SureLock, Invitrogen) at constant voltage of 30 V at 5°C for 60 min in NuPAGE transfer buffer with 10% methanol. After the transfer, the membrane was washed for 5 min in 50% methanol; then stained for 1 min in 0.1% Coomassie Blue R-250, 50% methanol, and 5% acetic acid; and then destained in 50% methanol until bands were visible. The membrane was left to air dry, and the protein bands were cut out and submitted for N-terminal Edman sequencing on Procise protein sequencing system 492A (PE Applied Biosystems at Jožef Stefan Institute, Ljubljana, Slovenia). Edman chemistry using pulsed-liquid blot was performed for preparation of phenylthiohydantoin amino acid derivatives. Analysis of the derivatives was performed on high-performance liquid chromatography (HPLC) system 140C (PE Applied Biosystems) using RP C18 column Spheri-5 (5 μm, 220 mm by 2.1 mm) (Brownlee).
7
Purified KpDyP Protein Analysis
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Purified free KpDyP was subjected to size exclusion analysis on a Superdex 200 Increase 10/300 GL column. Fractions were then analyzed via native PAGE in an Invitrogen XCell SureLock using NativePAGE 3–12% bis-tris mini protein gels, 1X NativePAGE Running Buffer, and NativeMark Unstained Protein Standard from Fisher Scientific (USA) with 1× running buffer made from 10× Tris/Glycine Buffer from Bio-Rad Laboratories, Inc. (USA). Approximately 10 μg of protein was loaded per well. NativePAGE gels were run at 150 V for 1 h, then 250 V for 2.5 h at 4 °C. Gels were stained with ReadyBlue Protein Gel Stain from Sigma-Aldrich (USA) and imaged and analyzed on a ChemiDoc Imaging System by Bio-Rad Laboratories, Inc. (USA).
8
Insulin Release Analysis via Electrophoresis
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Human insulin (control), a protein ladder (See Blue® Plus2 Pre-stained Protein Standard, Novex™ Thermo Fisher Scientific, UK), insulin released from both NP and PVA-borate hydrogels were placed in the wells of a NUPAGE® Bis-Tris 12% gel (Invitrogen, Thermo Fisher Scientific, UK) using a mini-cell electrophoresis system (X-cell Surelock™, Invitrogen, Thermo Fisher Scientific, UK). Peptide samples (10 μl) were vortexed with 2.0 μl Laemmli buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) and heated at 100 °C for 10 min. Electrophoresis was run at 200 V (~ 100 mA) until samples reached the bottom of the gel. A fixative solution of Coomassie blue dye was used to stain the gel followed by addition of a destaining solution of methanol/acetic acid/water (5:4:1 v/v), which visualised the peptide bands. GelDoc-It™ (UVP, Cambridge, UK) was used to photograph the gel and record band positions.
9
Native PAGE Analysis of Encapsulin Shells
10
Protein Sample Preparation and Electrophoresis
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Samples were mixed 1:4 with NuPAGE® LDS sample buffer (Invitrogen) and either heated to 95 °C for 5 min or, when analyzing ACKR2 expression, incubated at room temperature for 10 min (44 (link)). Samples were mixed 1:10 with NuPAGE® sample reducing agent (Invitrogen). 10–20 μl of each sample was loaded onto a precast NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen) in a vertical electrophoresis tank (Xcell Surelock; Invitrogen) filled with NuPAGE MES SDS running buffer (Invitrogen). Novex® Sharp prestained protein standard (Invitrogen) was run alongside samples for size determination. Electrophoresis was performed for 1–2 h at 150 V.
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