HPK in storage buffer (20 mM HEPES [pH 7.45], 150 mM NaCl, 10% glycerol) was distributed into 150-μl aliquots at 1.33 μg/μl, and the pHs were adjusted by mixing with 350 μl of storage buffer at titrating pH levels for 30 min at 25°C or 37°C (0.4 μg/μl final protein concentration). Final pH values were confirmed using an Oakton pHTestr 50S (Cole-Parmer). Each sample underwent size filtration to retain HPK oligomers as previously established (21 (link),44 (link)), and was sustained in its own pH buffer during application to spin columns that sufficiently retain HPK oligomers if present (Amicon Ultra 0.5-ml centrifugal filters, 100K NMWL). Each retentate (20 μl) was directly loaded on a Mini-protein TGX stain-free gel (4–15%; Bio-Rad); the 10% glycerol in the storage-incubation buffer was sufficient for loading the protein on the gel. After electrophoresis using standard native electrophoresis buffer (25 mM Tris, 192 mM glycine, pH 8.3) at 200 V for 35 min, the gels were visualized using a ChemiDoc MP imaging system (Bio-Rad). The bands were additionally visualized by Coomassie staining according to standard procedures.
Mini protein tgx stain free gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protein TGX Stain-Free Gel is a precast polyacrylamide gel used for protein separation and analysis in electrophoresis. It is designed to provide fast, efficient, and consistent separation of proteins without the need for traditional staining procedures.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Trans blot turbo midi pvdf transfer pack, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab184699, by Abcam (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ab201015, by Abcam (1 mentions)
Dc protein assay kit 1, by Bio-Rad (1 mentions)
Chemidoc md imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Pvdf membrane, by Macherey-Nagel (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Prism 5, by GraphPad (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Ube2c, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
9 protocols using mini protein tgx stain free gel
1
pH-Dependent Oligomerization of HPK
2
Western Blot Analysis of Protein Expression
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Antibodies were purchased as follows: IGF-II Receptor/CI-M6PR (D3V8C) and β-actin (# 8H10D10) (Cell Signaling Technology, Danvers, MA, USA), α-Gal-A (#GTX101178) (GeneTex, Irvine, CA, USA). Whole-cell extracts (WCEs) were prepared in radioimmunoprecipitation (RIPA) buffer. Protein concentrations were determined using the BCA Protein Assay Kit (ThermoFisher, Rockford, IL, USA). Thirty micrograms (30 μg) of WCE were separated on mini protein TGX stain-free gel (Bio-Rad, Hercules, CA, USA) and electroblotted using the Trans-Blot® Turbo™ Midi PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). Membranes were diluted with antibodies (1:1000 dilutions) in 5% BSA, 1 × TBS, 0.1% Tween20, and gently shaking overnight at +4 °C. The ChemiDocTM MP Imaging system (Bio-Rad) was used to visualize and quantitate optical density (IOD). The IODs of bands of interest were normalized to the loading control actin used in the same blot [8 (link)], and the normalized value of the controls was set to 1 for a comparison between separate experiments.
3
Immunoblotting of MAPK Pathway
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A total of 1 × 106 cells were lysed with buffer (150 mM sodium chloride; 1.0% Triton X-100; 50 mM Tris pH 8.0); 30 μg of protein was loaded into wells of Mini-protein TGX stain free gel (Bio-Rad, 456 8094). After transfer of proteins to polyvinylidene difluoride membrane, it was incubated overnight with anti-ERK (Abcam, ab184699), anti-pERK (Abcam, ab201015), anti-pRSK (Abcam, ab62324), or anti-Actin as loading control (Abcam, ab115777) antibodies in iBIND (ThermoFisher) according to the manufacturer’s protocol using iBIND buffers. Membranes were imaged with Chemidoc (Bio-Rad), and the intensity of the bands was calculated in ImageStudioLite.
4
Quantitative Western Blot Analysis
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Whole-cell extracts (WCE) were prepared in radioimmunoprecepitation (RIPA) buffer. Protein concentrations were determined using the BCA Protein Assay Kit (ThermoFisher Scientific, Rockford, IL, USA). 30–40 μg of WCE were separated on mini protein TGX stain free gel (Bio-Rad, Hercules, CA, USA) and electroblotted using the Trans-Blot Turbo Midi PVDF Transfer Packs (Bio-Rad). The ChemiDoc MP Imaging system (Bio-Rad) was used to visualize and quantitate optical density (IOD) for each band. The IODs of bands of interest were normalized to the loading control and used in the same blot: e.g. Ponceau staining bands, α-tubulin or b actin and the normalized value of the controls was set to 1 for comparison between separate experiments.
5
O-Acetylation Kinetics of Xylan by AtXOAT1
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To verify the positional specificity of AtXOAT1-cat, the reaction of O-acetylation of Xyl6 (Amicon®, Merck Millipore Ltd., Ireland) . The absence of AtXOAT1 in the filtrate, and its complete retention in the filter device, was confirmed by both SDS-PAGE using a precast Mini-PROTEIN® TGX Stain-Free™ Gel (Bio-Rad Laboratories, Inc.) and by measuring the direct absorbance at 280 nm using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). The filtrate was immediately transferred to the NMR spectrometer, and the signals that correspond to the CH 3 of the O-acetyl substituents at different positions were monitored by arrayed 1 H NMR according to the following process. Data were recorded at 298 K with an Agilent-NMR spectrometer equipped with a 5-mm NMR cold probe operating at 600 MHz. Each 1D 1 H spectra consists of 16 transients, and were acquired with water presaturation every 30 min for 14 hrs overnight. The spectral array generated represents the reaction progress. Samples were never subjected to lyophilization.
6
Western Blot Protein Analysis Protocol
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Cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, in 1 × TBS) with protease inhibitors (25 µg/ml chymostatin, 25 µg/ml leupeptin, 25 µg/ml antipain hydrochloride, 25 µg/ml pepstatin A). Protein concentration was measured using DC™ Protein Assay Kit I (Bio-Rad, 5000111) and 10–15 µg of protein was loaded on Mini-Protein TGX Stain-Free gels (Bio-Rad, 1610181, 1610183, 1610185, and 5678094) and run at 120 V in 1 × SDS-Page running buffer. After running, gels were activated using the ChemiDoc MD Imaging System (Bio-Rad) and transferred onto 0.45-µm Low Fluorescence PVDF membranes (Bio-Rad, 1704274). Membranes were blocked with 5% skim milk in 0.1% Tween-20 in TBS (TBS-T) for 45 min and subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. Membranes were washed 3 × in TBS-T and incubated with secondary antibodies at room temperature for 45 min. Membranes were washed 3 × with TBS-T and incubated with Clarity Western ECL substrate (Bio-Rad, 1705061) and imaged using a ChemiDoc MD Imaging System (Bio-Rad).
7
Grx2 Protein Quantification in Patients
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Determination of total protein was done using Bradford-reagent (Bio-Rad Laboratories, Hercules, CA, USA). SDS-PAGE and Western blot for Grx2 in patient samples were run using the mini-Protein TGX stain-free gels (4–20%, Bio-Rad), and PVDF membranes (Macherey & Nagel) according to manufacturer’s instructions. Following semi-dry blotting, membranes were blocked with 5% milk powder and 1% BSA in TBS, supplemented with 0.05% Tween 20, for 1 h at room temperature and were incubated with primary antibody overnight at 4°C. Following extensive washing with TBST, membranes were incubated with horseradish peroxidase conjugated secondary antibodies (Bio-Rad). Western blots were finally developed by the enhanced chemiluminescence technique. For band quantification ImageJ 1.5.2a (NIH, USA) was used and band intensity calculated by Graph Pad Prism 5 (La Jolla, CA, USA).
8
Quantifying UBE2C Protein Expression
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Equal amounts of protein (10 µg) were loaded into Mini-Protein TGX stain-free gels (Bio-Rad) and separated using gel electrophoresis. Proteins were transferred to polyvinyl-fluoride membrane and 5% skimmed milk in Tris-buffered saline-Tween20 was utilized to block unspecific binding. Primary antibody incubation was performed at room temperature for overnight (UBE2C at dilution 1:500; #14234S; Cell Signaling Technology Inc., Danvers, MA, United States). Secondary antibody incubation was carried out at room temperature for 1 h (goat anti-rabbit IgG at dilution 1:10,000; #111-035-144, Jackson ImmunoResearch, West Grove, PA, United States). Protein bands were visualized using Enhanced Chemiluminescence detection kit (Amersham ECL reagent; GE Healthcare, Barrington, IL, United States). Protein quantification was performed with Image Lab software (version 6.0, Bio-Rad) by normalizing UBE2C band intensities to amount of total protein in corresponding lane utilizing stain-free technology (Gürtler et al., 2013 (link)).
9
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer (89900, Thermo) supplemented with a protease inhibitor cocktail (Roche). Protein concentrations were determined by a Detergent Compatible Bradford Assay kit (23246, Thermo). Calibrated samples were diluted with 4x Laemmli sample buffer (Bio-Rad), and equal amounts of total protein were separated in Mini-protein TGX stain-free gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-blot Turbo Transfer System (Bio-Rad). The membranes were blocked with 5% nonfat milk in TBST, incubated with primary antibodies overnight at 4 °C, washed three times with TBST at room temperature, incubated with HRP secondary antibodies, and imaged using the Bio-Rad Chemidoc imaging system. All antibodies and dilution information used in this study are listed in Supplementary Data 8 .
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