Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride or nitrocellulose membranes (Thermo Fisher Scientific). Membranes were blocked either with 5% bovine serum albumin (BSA, Sigma-Aldrich) or 5% milk in Tris-buffered saline (TBS). Proteins were analyzed with rabbit antibodies against myelin basic protein (Mbp, 1:400, AB980, Thermo Fisher Scientific), microtubule-associated protein 2 (Map2, 1:500, ab24640, Abcam, Cambridge, UK) and β-Tubulin (1:4000, ab6046, Abcam) in 5% milk (for Mbp) or 5% BSA (for Map2 and β-Tubulin) in TBS-Tween 20 (TBS-T, Sigma-Aldrich) overnight at 4 °C. Horseradish peroxidase-coupled donkey anti-rabbit (1:1000, GE Healthcare Life Science, Piscataway, NJ, USA) antibody was used as a secondary antibody and incubated with the membranes for 1 h at room temperature. Binding was detected using the chemiluminescent Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) on a Chemidoc XRS+ system (Bio-Rad). Pixel summation of individual bands was performed with ImageJ Software (NIH, Bethesda, MD, USA). The ratio between Mbp/Map2 and β-Tubulin was calculated for each animal.
4 to 20 gradient gel
Manufactured by Bio-Rad
Sourced in United States
The 4 to 20% gradient gel is a laboratory equipment used for electrophoresis. It is designed to separate proteins based on their molecular weight. The gel consists of a polyacrylamide matrix with a gradually increasing concentration of acrylamide from 4% to 20%, which creates a gradient that allows for the separation of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Quick start bradford reagent, by Bio-Rad (2 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Ab6046, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Ab24640, by Abcam (1 mentions)
Tbs t, by Merck Group (1 mentions)
Horseradish peroxidase coupled donkey anti rabbit, by GE Healthcare (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Mammalian cell lysis kit, by Merck Group (1 mentions)
Amersham ecl prime western blotting reagent, by GE Healthcare (1 mentions)
Gfp trap a beads, by Proteintech (1 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Ab167453, by Abcam (1 mentions)
10 protocols using 4 to 20 gradient gel
1
Western Blot Analysis of Myelin and Cytoskeletal Proteins
2
Quantitative Analysis of Protein Expression in Postmortem Monkey Brains
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Preparation and immunoblotting were performed as previously described (40 (link)). Frozen, powdered samples from postmortem monkey brains were sonicated in 1% SDS and boiled for 10 min. Aliquots (1 μl) of the homogenate were used for the protein determination by a Bio-Rad Protein Assay kit (Bio-Rad). Equal amounts of total proteins (30 μg) for each sample were loaded on precast 4 to 20% gradient gel (Bio-Rad). Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes through Trans-Blot Turbo System. Membranes were then immunoblotted overnight using the following primary antibody: anti-RasGRP1 (1:1000; no. MABS146), anti-GFAP (1:1000; no. sc-33673), anti-PDE2A (1:1000; no. 55306-1-AP), anti-GAD1/2 (1:1000; no. ABN904), and anti-TH (1:2000; no. MAB318). Blots were then incubated in horseradish peroxidase–conjugated secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (GE Healthcare) and quantified by Quantity One software (Bio-Rad). Optical density values were normalized to GAPDH (1:1000; no. SC-32233) for variation in loading and transfer. Normalized values were then averaged and used as dependent variable. Statistical analysis was performed by using Mann-Whitney test.
3
Gβ Protein Induction Kinetics
4
Western Blot Analysis of GRP94 in EVs
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Twenty microliters of WJ-MSCs, lysed using the mammalian cell lysis kit from Sigma-Aldrich (St. Louis, MO, USA), and EV were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad) to analyze the expression of the chaperone GRP94. Separated proteins were transferred onto nitrocellulose membranes (Thermo Fisher Scientific), blocked with 5% milk, and analyzed with a rabbit antibody against GRP94 (1:500; Cell Signalling Technology, Danvers, MA, USA). As secondary antibody, horseradish peroxidase (HRP)-coupled donkey anti-rabbit (1:1,000; GE Healthcare Life Sciences) was used. Binding was detected by chemiluminescence using Amersham ECL Prime Western blotting reagent (GE Healthcare Life Sciences) on a Chemidoc XRS+ system Bio-Rad.
5
Immunoprecipitation and Mass Spectrometry of Plant Effectors
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Total proteins were extracted from N. benthamiana leaves 3 days after agroinfiltration of GFP::AVRcap1b or GFP::PexRD54 [74 (link)] and subjected to immunoprecipitation using GFP_Trap_A beads (Chromotek, Munich, Germany), as described previously [50 (link),97 (link),100 (link)]. PexRD54 was included as a control, as it is also a large P. infestans RxLR effector and extensive studies suggests that its role is likely independent of the NRC network [74 (link),75 (link)]. Final OD600 for each protein is indicated in S13 Table . Immunoprecipitated samples were separated by SDS-PAGE (4% to 20% gradient gel, Biorad, United Kingdom) and stained with Coomassie brilliant Blue G-250 (SimplyBlue Safe stain, Invitrogen). Enriched protein samples were cut out of the gel (approximately 5 × 10 mm) and digested with trypsin. Extracted peptides were analysed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with the Orbitrap Fusion mass spectrometer and nanoflow-HPLC system U3000 (Thermo Fisher Scientific, UK) [48 (link),50 (link)]. A total of 3 biological replicates for each sample type were submitted.
6
Protein-Protein Interaction Assay in HEK293 Cells
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The following constructs were cotransfected in a 10-cm dish containing 30 to 40% confluent HEK293 cells using either Lipofectamine (Life Technologies) or jetPRIME reagent (Polyplus-transfection) following the manufacturer’s instructions: GFP-FAM161A with mCherry-POC5, mCherry-POC5 alone, GFP–Centrin-2 with mCherry-FAM161A, and GFP-POC5 with mcherry-POC1B. After 24 hours of expression, cells were processed according to the GFP-Trap Kit (Chromotek) instructions using radioimmunoprecipitation assay (RIPA) buffer for the lysis and incubating the lysate with GFP-Trap magnetic beads for 2 hours at 4°C on a rotating wheel. Samples were run on a 4 to 20% gradient gel (Bio-Rad) before transfer on polyvinylidene difluoride (PVDF) membrane using the iBlot system (Invitrogen/Thermo Fisher Scientific). Membranes were either incubated with primary antibodies against mouse GFP (1:700; Abcam, ab1218) or rabbit mCherry (1:500; Abcam, ab167453), followed by goat anti-mouse horseradish peroxidase (HRP) (1:1000 from a 50% glycerol stock; Thermo Fisher Scientific, 31431) or goat anti-rabbit HRP (1:1000 from a 50% glycerol stock; Thermo Fisher Scientific, 31468) secondary antibodies. Blots were developed using Amersham films (GE Healthcare, 28906836).
7
Quantitative Western Blot Protein Analysis
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Total cellular protein was prepared as described previously [30 (link)]. Protein concentration was measured using Quick Start Bradford Reagent (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard. Protein aliquots were stored at −80°C until use. For Western blot analysis, 20 μg of total cellular protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad); then the blot was transferred onto a polyvinylidene difluoride membrane (Life Technologies, Carlsbad, CA). Blocking, 1st and horseradish-peroxidase-conjugated 2nd antibody reactions and washing were conducted as previously described [28 (link)]. The chemiluminescence signals were visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK) and ChimiDoc XRS Plus ImageLab System (Bio-Rad). The 1st and 2nd antibodies were purchased from common suppliers.
8
VHH-MG Fusion Protein Analysis
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VHH-MG fusions (crude/purified) were analyzed on a commercial 4 to 20% gradient gel (Bio-Rad) under reducing conditions and stained with Coomassie G-250 following the manufacturer's instructions. For western blotting, proteins were transferred onto a polyvinylidene difluoride membrane (PVDF) using the semidry transfer method (Bio-Rad). Blotted membrane was blocked overnight with 2% skimmed milk in PBST (PBS + 0.1% (v/v) Tween-20) at 4 o C. Thereafter, the membrane was probed with anti-mouse IgG (GE Healthcare, NXA931) conjugated to horseradish peroxidase (HRP), diluted 1:2000 in blocking solution and incubated for 1 h at RT. The membrane was then washed three times with PBST, and bands were visualized by adding HRP substrate (WesternBright ECL, Advansta). The membrane was imaged using a ChemiDoc MP imaging system (Bio-Rad) and analyzed by ImageLab software (Bio-Rad).
9
NFX1-123 Protein Expression Profiling
10
Quantitative Western Blot Analysis of Proteins
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Total cellular protein was prepared as described previously [29 (link)], and the protein concentration was measured using Quick Start Bradford Reagent (Bio-Rad, Hercules, CA, USA). Twenty micrograms of protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) in a 4 to 20% gradient gel (Bio-Rad), and the blot was transferred onto a polyvinylidene difluoride membrane by iBlot 2 (Life Technologies, Carlsbad, CA, USA). After blocking with 0.2% nonfat dry milk (Cell Signaling Technology, Danvers, MA, USA) in Tris-buffered saline (Takara Bio), the membrane was incubated with the primary antibodies (anti-TPD52 antibody (1/1,000 dilution; Abcam, Branford, CT, USA), anti-TPD54 antibody (1/1,000 dilution), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1/10,000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and the horseradish-peroxidase-conjugated secondary antibody (GE Healthcare UK Ltd., Buckinghamshire, UK)) as described previously [29 (link)]. The protein bands were visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare) and a Chemidoc XRS Plus ImageLab System (Bio-Rad).
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