20 μg of hippocampal total protein in 2× loading buffer (BioRad, #1610738, without SDS) was loaded onto a 5.5% native polyacrylamide gel (1.5 M Tris-HCl, Resolving Gel buffer, ProtoGel, 30% acrylamide, 10% APS, TEMED, without SDS) with native unstained protein marker (Invitrogen, LC0725). Gels were run at 120 V for 2 h followed by a transfer onto PVDF membrane in Tris-glycine-0.1% SDS transfer buffer at 200 mA/16 V overnight at 4 °C. Membranes were blocked in 5% nonfat dry milk in tris-buffered saline (TBS) and 0.1% Tween-20 and incubated overnight in mouse anti-HSP90 (H9010, Stressmarq SMC-107, 1:2000), rat anti-HSC70 (Enzo, ADI-SPA-815, 1:2000) or mouse anti-HOP (Enzo, SRA-1500, 1:2000). Membranes were incubated in respective horseradish peroxidase conjugated secondary antibodies (Thermo-Fischer) at room temperature for 1 h and developed for image quantification. Chemiluminescent signals were visualized using chemiluminescence detection system (Supersignal, Pierce). Blots were imaged with the Chemidoc Imager (BioRad). Optical density (OD) of protein bands were measured using ImageJ (National Institutes of Health). OD of HMW bands (between 480 and 720kD for HSP90 and HSC70; between 250 and 300kD for HOP) were normalized to SDS PAGE measurements of associated monomeric protein and actin OD levels (described below).
Lc0725
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States, United Kingdom
The LC0725 is a liquid chromatography system designed for analytical purposes. It is capable of separating and analyzing complex mixtures of chemical compounds. The core function of the LC0725 is to provide reliable and accurate chromatographic data for various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Discovermp software, by Refeyn (6 mentions)
Acquiremp software, by Refeyn (3 mentions)
Onemp instrument, by Refeyn (2 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Adi spa 815, by Enzo Life Sciences (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sra 1500, by Enzo Life Sciences (1 mentions)
Xcell mini cell system, by Thermo Fisher Scientific (1 mentions)
Bn1002box, by Thermo Fisher Scientific (1 mentions)
Coomassie g 250, by Thermo Fisher Scientific (1 mentions)
Bn2007, by Thermo Fisher Scientific (1 mentions)
Bn2003, by Thermo Fisher Scientific (1 mentions)
Nativepage 4 16 bis tris gel, by Thermo Fisher Scientific (1 mentions)
G250 additive, by Thermo Fisher Scientific (1 mentions)
50 mm microscope coverslips, by Thermo Fisher Scientific (1 mentions)
Gbl103250, by Merck Group (1 mentions)
Acquiremp, by Refeyn (1 mentions)
Discovermp, by Refeyn (1 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast polyacrylamide gel, by Bio-Rad (1 mentions)
14 protocols using lc0725
1
Native PAGE Analysis of Hippocampal Chaperones
2
Blue Native PAGE Analysis of CICs
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CICs were resolved using BN-APAGE system as previously described (34 (link)). Samples and unstained native markers (catalog no. LC0725, Invitrogen) were mixed with the sample buffer [0.5% Coomassie blue G-250 and 50 mM ε-aminocaproic acid in 10 mM bis-tris (pH 7.5) at final concentration] just before use, and electrophoresis was performed using 1× anode buffer [50 mM bis-tris HCl (pH 7.0)] and 1× cathode buffer [50 mM tricine, 15 mM bis-tris, and 0.0015% G-250 (pH 7.0)] for at ~18 hours using relatively low voltage and low current (e.g., 15 to 20 V, ~2 mA, for four gels).
3
Visualization of Native Canola Protein Profiles
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The native protein profiles of duplicate canola meal samples were visualised by gel-electrophoresis following manufacturer’s instructions. In brief, sample (~10 µg CP per µL) was added to 2.5 µL NativePAGETM sample buffer (4×), 1 µL NativePAGETM 5% G-250 sample additive, and made to 10 µL with deionised H2O. The sample and NativeMarkTM unstained protein standard (5 µL, LC0725, Invitrogen, Mulgrave, VIC, Australia) were loaded onto a NativePAGETM 4–16% gradient precast bis-tris (10 × 10 cm2) gel in a Novex Xcell mini cell system (Invitrogen, Mulgrave, VIC, Australia). The running buffer contained 50 mM BisTris, 50 mM tricine, pH 6.8, and the sample buffer contained 50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% w/v glycerol, and 0.001% Ponceau S, pH 7.2. The upper (inner) buffer chamber contained cathode buffer (200 mL: 10 mL NativePAGETM running buffer 20×, 10 mL NativePAGETM cathode additive 20×) and the lower (outer) buffer chamber contained anode buffer (600 mL: 50 mL NativePAGETM running buffer 20×, 950 mL deionised H2O). Electrophoresis was performed at 150 V for 110 min. The native proteins were visualised by incubating the gel in 40% methanol and 10% acetic acid for 25 min, Coomassie Brilliant Blue R-250 solution (0.02% in 30% methanol and 10% acetic acid) for 25 min, and 8% acetic acid on an orbital shaker at RT overnight.
4
Purification and Native-PAGE of Spartin Proteins
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Spartin proteins including HsSpartin, CeSpartin and CtSpartinL were expressed in Expi293 cells, and purified using buffer A (50 mM HEPES, 500 mM Nacl, pH8.0, 1 mM TCEP, 10% glycerol) using anti-FLAG resin. Proteins were quantified using BSA standards in SDS-PAGE. Proteins (1–2 μg) together with BSA (1 μg) were loaded onto native PAGE 4%−16% Bis-Tris gel (BN1002BOX, invitrogen) for electrophoresis at 4C using NativePAGE running buffer and NativePAGE Cathode buffer (contains 0.02% Coomassie G-250, BN2007, invitrogen). NativeMark unstained protein standards (LC0725, invitrogen) and NativePAGE sample buffer (BN2003, invitrogen) were used. Protein gels were fixed and destained according to manufacturer direction. Distinct from native gels in the lipid-co-migration assay (above), where samples migrate according to their charge/mass ratio, here the Coomassie dye in the cathode buffer coats protein sample, which consequently migrates according to MW (28 (link)).
5
Oligomeric State Analysis of DdMCU-NTD
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The oligomeric
state of DdMCU-NTD was first examined by chemical cross-linking using
glutaraldehyde (Sigma). The reaction volume of 50 μL consisted
of 0.5 mg/mL protein, 25 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, and
various concentrations of glutaraldehyde (0.0015, 0.015, and 0.15%,
v/v). The reaction was stopped after 2 min by adding 100 mM Tris–HCl
(pH 8.0). The samples were incubated for another 5 min. The reaction
mixtures were analyzed using SDS-PAGE gel. Blue native gradient gel
was generated with a NativePAGE 4–16% Bis–Tris Gel (Invitrogen,
16041980). Two micrograms of DdMCU-NTD (in the absence and presence
of 20 mM CaCl2) was mixed with NativePAGE sample buffer
and G-250 additive (Invitrogen), then analyzed by blue native gel.
The electrophoresis was performed at 150 V in 1× NativePAGE anode
buffer and 1× NativePAGE cathode buffer A for 70 min. The NativeMark
unstained protein standard (Invitrogen #LC0725) was used as a reference
marker.
state of DdMCU-NTD was first examined by chemical cross-linking using
glutaraldehyde (Sigma). The reaction volume of 50 μL consisted
of 0.5 mg/mL protein, 25 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, and
various concentrations of glutaraldehyde (0.0015, 0.015, and 0.15%,
v/v). The reaction was stopped after 2 min by adding 100 mM Tris–HCl
(pH 8.0). The samples were incubated for another 5 min. The reaction
mixtures were analyzed using SDS-PAGE gel. Blue native gradient gel
was generated with a NativePAGE 4–16% Bis–Tris Gel (Invitrogen,
16041980). Two micrograms of DdMCU-NTD (in the absence and presence
of 20 mM CaCl2) was mixed with NativePAGE sample buffer
and G-250 additive (Invitrogen), then analyzed by blue native gel.
The electrophoresis was performed at 150 V in 1× NativePAGE anode
buffer and 1× NativePAGE cathode buffer A for 70 min. The NativeMark
unstained protein standard (Invitrogen #LC0725) was used as a reference
marker.
6
Single Particle Mass Analysis of Viral Capsids
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The MP video files were processed using the DiscoverMP software (Refeyn, Oxford, UK), as previously described [35 (link)]. The threshold parameter value of 5 was used in the analysis (Supplementary Material, Fig. S2 ). MP contrast distributions were plotted as Kernel Density Estimates (KDE) or histograms. For all plots, the KDE contrast bandwidth and the histogram bin size were both set to 0.003 contrast units. To obtain information on the contrast distribution species, the MP histograms were fit with Gaussian peaks. For each fitted species, the best fit Gaussian peak position and area represent their average contrast value and their number fraction, respectively. Molecular mass of the empty capsids was estimated from the MP contrast distribution by applying the Contrast-to-Mass (CTM) calibration obtained using an unstained protein ladder sample (LC0725, Thermofisher, Wattham, MA) [35 (link)].
7
Biomass Quantification of Q-beta Conjugates
8
Mass Distribution Analysis of RrA Samples
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The mass distributions of RrA samples were assessed using a Refeyn TwoMP instrument (Refeyn, Oxford, UK). The measurements adhered to the standard protocol as outlined in Wu & Piszczek (2021 (link)). In short, sample chambers were assembled by positioning silicon gasket wells onto 24 mm × 50 mm coverslips. Initially, the sample wells were loaded with predetermined volumes of the standard buffer, having been passed through a 0.22 μm syringe filter. Solutions of RrA were clarified through centrifugation, with the enzyme concentrations determined based on the absorbance at 280 nm and on theoretical extinction coefficient at this wavelength. Data were gathered at two distinct concentrations, 25 and 65 nM, in the standard buffer. A one‐minute video was recorded employing the AcquireMP software and subsequent analysis was performed using DiscoverMP (Refeyn, Oxford, UK). Before each RrA measurement, a mixture of well‐characterized proteins (unstained protein ladder, LC0725, ThermoFisher Scientific, Waltham, MA) was measured under identical conditions. This measurement was used for reliable calibration of molecular weights.
9
Mass Distribution Analysis of ASNase
10
Native PAGE Protocol for Protein Analysis
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Proteins were prepared (9 μl) at the required concentration (10 to 20 μM) in 10 mM tris-HCl (pH 8.0), 5 mM EDTA, and 1% β-OG buffer, to which 0.45 μl of 5% G250 (Thermo Fisher Scientific, BN2004) and 3 μl of 4× NativePAGE sample loading buffer (Thermo Fisher Scientific BN2003) were added. Running buffer (1×) was made from 20× stock of NativePAGE running buffer (Thermo Fisher Scientific, BN2001), and 1× light blue cathode buffer was made from 20× stocks of NativePAGE running buffer and Native PAGE cathode additive (Thermo Fisher Scientific, BN2002). NativePAGE Novex 4 to 16% bis-tris protein gel (Thermo Fisher Scientific, BN1002BOX) and buffer solutions were cooled to 4°C before loading samples (8 μl) and native mark unstained protein ladder (Thermo Fisher Scientific, LC0725). Gels were run at 4°C at 150 V for 1 hour, followed by 1 hour at 250 V. Gels were stained as described above for SDS-PAGE gels, with staining duration increased to 1 hour.
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