For Western blot analysis, the supernatants were thawed on ice and precipitated by 10 μl Strata clean resin (Agilent, Waldbronn). For SDS PAGE, the precipitated proteins were dissolved in 50 μl sample loading dye (3 X Laemmli beta‐mercaptoethanol), boiled for 7 min and stored on ice. 10 μl of each sample was loaded on 12% SDS‐PAGE. After gel electrophoresis proteins were transferred onto nitrocellulose membrane (Protran, GE, Frankfurt am Main) using Trans Blot device (Bio‐Rad, Munich) at 350 V for 40 min. Membranes were then blocked overnight with RotiBlock blocking reagent (Roth, Karlsruhe). Membranes were incubated with primary antibody (anti‐strep‐tag rabbit IgG, Abcam) for 1 hr and subsequently with secondary antibodies (anti‐rabbit IgG goat IgG alkaline phosphatase conjugated, Sigma) for 1 hr each at room temperature under gentle shaking. Detection was carried out using BCI/NBP (Sigma, Munich); blots were scanned by Epson scanner.
Protran
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany
Protran is a lab equipment product offered by GE Healthcare. It is designed for the purpose of protein and nucleic acid transfer and separation. The core function of Protran is to facilitate the transfer and separation of these biological molecules for various analytical and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (3 mentions)
Odyssey classic instrument, by LI COR (3 mentions)
Dot blot vacuum device, by Cytiva (3 mentions)
Whatman, by Cytiva (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Nupage bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Amersham hybond, by GE Healthcare (2 mentions)
Ab105767, by Merck Group (2 mentions)
Ab137027, by Abcam (2 mentions)
Ab290, by Abcam (2 mentions)
Labtek 2 chambered coverslips, by Thermo Fisher Scientific (2 mentions)
Mab1501, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Image studio lite version 5, by LI COR (2 mentions)
Irdye 680rd, by LI COR (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Odissey systems, by LI COR (2 mentions)
β actin, by Merck Group (2 mentions)
Protein assay dye reagent, by Bio-Rad (2 mentions)
Strataclean resin, by Agilent Technologies (1 mentions)
35 protocols using protran
1
Western Blot Analysis of Strep-Tagged Proteins
2
Western Blotting of ESCRT Proteins
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Cells were seeded and transfected as described above in LabTek II chambered coverslips (ThermoScientific). Protein extracts were separated on 4-12% NuPAGE Bis-Tris gradient gels (Life Technologies) and transferred to Nitrocellulose (Protran, GE Healthcare) or PVDF membranes (Amersham Hybond, GE Healthcare). Western blotting was performed by standard methods using antibodies against CHMP4B (1:1000, Abcam, ab105767), actin (1:30000, Merck Millipore, MAB1501), VPS4B (1:500, Abcam, ab137027), GFP (1:5000, Abcam, ab290) and GAPDH (1:2500, Abcam, ab9485).
3
Western Blot Analysis of Exosome Markers
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HCT116 cells grown in 24-well plates were pelleted by centrifugation and resuspended in ∼50 μl of 2× protein loading dye (75 mM Tris–HCl, pH 6.8, 1.25 mM EDTA, 20% glycerol, 2.5% SDS, 0.125% bromophenol blue, and 50 mM DTT). 10 μl of each sample was loaded on a 1.5 mm denaturing SDS 13% polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Protran; GE Healthcare) and probed with monoclonal primary antibodies recognising EXOSC8, EXOSC9, p53, karyopherin β1, or GAPDH, diluted in PBS-Triton X-100 (0.1%, vol/vol) containing 2% non-fat dried milk (wt/vol) (Marvel). An IRDye-labelled secondary antibody (LI-COR) was used, and membranes were visualised using the Odyssey LI-COR system (LI-COR). Quantification was performed using ImageQuant software (GE Healthcare) and quantified protein levels were normalised to levels of the loading control, karyopherin. The following antibodies were used: α-EXOSC8 (mouse, (H-8) sc- 393027; Santa Cruz), dilution 1:1,000; α-EXOSC9 (mouse, (D-6) sc-271815; Santa Cruz), dilution 1:1,000; α-p53 (mouse, (DO-1) sc-126; Santa Cruz), dilution 1:500; α-karyopherin β1 (mouse, (H-7) sc-137016; Santa Cruz), dilution 1:2,000; α-GAPDH (mouse, (0411) sc-47724; Santa Cruz), dilution 1:10,000; and secondary antibody Donkey α-Mouse 800CW LI-COR (926-32212), dilution 1:10,000.
4
Quantitative Western Blot Analysis of Protein Levels
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Brain extracts were prepared from wt and ko mice in parallel and comparisons of protein content were only made between extracts prepared in parallel, which were separated next to each other on the same SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare Protran, 0.45 μm). All wt/ko data pairs are from independent biological samples. Comparing the data from different animals and independent preparations requires a normalisation and thus wt values were defined as 100%. The protein load was varied between 10 and 80 μg per lane to determine the linear protein/chemiluminescence signal ratio. Several proteins of different molecular masses were detected on one western-blot nitrocellulose membrane. This served as internal control for protein isolation and detection by the ECL luminescence detection kits PICO, NANO, FEMTO (Pierce-ThermoScientific, Karlsruhe, Ger), recorded with a Fuji LAS 1000 (Fujifilm Corp., Düsseldorf, Ger) camera system. Protein determination: Bradford-assay (BioRad, Munich, Ger) (Supplementary Material ).
5
Quantitative Immunoblotting Protocol
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Sample preparation for quantitative immunoblotting was done as described before [88 (link)]. Samples of 20 μg of protein in 2× Laemmli buffer were heated at 90 °C for 3 min and then separated in 6% tris–glycine polyacrylamide gels, using Precision Plus Protein™ All Blue Standards as size marker. Transfer to nitrocellulose membranes (Protran, GE Healthcare) was done at 20 V over night at 4 °C, with blocking in 5% BSA solution in 1× TBS-T for 1 h at room temperature (RT). Primary antibody incubation against RNF213 (Millipore, ABC1391, 1:1000) and HSP90 (Santa Cruz, sc-7947, 1:1000) occurred in 1× TBS-T solutions overnight at 4 °C. Fluorescence-labeled α-rabbit antibodies (1:15.000, IRDye 680RD, Li-Cor) were used as secondary antibodies. Fluorescence detection occurred with the Li-Cor Odyssey Classic Instrument and bands were densiometrically analyzed with Image Studio Lite, Version 5.2. (n = 3–4).
6
Spinal Cord Protein Extraction and Western Blot
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Spinal cord samples were homogenized in lysis buffer [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 5% glycerol, Phosphatase Inhibitor Cocktail Set 1 (Sigma), 0.2 mM sodium orthovanadate (Sigma), 10 mM sodium fluoride (VWR chemical), 2 mM 2-glycerophosphate (Chem-Impex International), 2 mM sodium pyrophosphate (Sigma), Complete™ Mini Protease Inhibitor Cocktail (Roche)] using an T10 Ultra-Turrax mechanical disperser (IKA). Homogenates were clarified by centrifugation at 15 000 g for 30 min at 4°C and soluble extracts were quantified by BCA assay (ThermoFisher Scientific). Protein extracts were diluted in Laemmli sample buffer and resolved on 7.5% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by transfer to 0.2 µm nitrocellulose membranes (Protran, GE Healthcare). Membranes were blocked in 5% milk in 1× TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature before incubation overnight at 4°C with primary antibodies and for 1 h with HRP-conjugated light chain-specific anti-IgG (Jackson Immunoresearch) in TBST. Proteins were visualized by enhanced chemiluminescence (ECL reagent, GE Healthcare) and imaged on an Amersham Imager 680 (GE Healthcare). Quantification of protein bands by densitometry was conducted using Image Studio™ Lite v5.2 software (LI-COR Biosciences).
7
Nitrocellulose Membrane-Based Filter Trap Assay
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For filter trap assays, native fractions were spotted onto nitrocellulose 0.2 µm membranes (Protran, GE) using a dot blot vacuum device (Whatman, Maidstone, UK). Nitrocellulose membranes were fixed for 30 min in PBS with PFA 0.4% (v/v) (Sigma-Aldrich, Saint-Louis, MO, USA) final concentration. After three washes with PBS, membranes were blocked with 5% (w/v) skimmed powder milk in PBS-Tween 0.5% (v/v) and probed with primary and secondary antibodies in PBS-Tween with 4% (w/v) BSA (Table 1 ). Immunoreactivity was whether visualized by chemiluminescence or infrared using Clarity ECL and Chemidoc (Biorad, Hercules, CA, USA) or Odissey systems (Li-Cor, Lincoln, NE, USA)) respectively.
8
Protein Extraction and SDS-PAGE Analysis
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Whole cell, cytoplasmic, nuclear or chromatin extracts were treated directly with 4x Laemmli buffer (0.2 M Tris–HCl, 8% (w/v) SDS, 40% glycerol, 20% (v/v) β-mercaptoethanol, 0.005% bromophenol blue), incubated at 95ºC for 5 min and sonicated. Samples were separated on mini-PROTEAN® TGX™ gels (Bio-Rad Laboratories) followed by transfer onto nitrocellulose membrane (Protran, GE Healthcare) and probed with antibodies.
9
Western Blot and Immunoprecipitation Protocols
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Protein levels were assessed as whole cell extracts, directly lysed, boiled and sonicated in 4× SDS Laemmli buffer (250 mM Tris–HCl pH 6.8, 8% SDS, 40% glycerol, 8% β-mercaptoethanol, 0.02% bromophenol blue). Samples were separated by SDS-PAGE using precast gels (Mini-PROTEAN TGX, BioRad), transferred onto nitrocellulose membranes (Protran, GE Healthcare) and probed with antibodies.
For immunoprecipitation, cells were trypsinized, washed in cold 1× PBS and centrifuged (1200rpm, 5 min). Pellets were lysed in 5 volumes lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, 100 U RNase inhibitor, 1× protease/phosphatase inhibitor cocktails, Roche) for 20 min on ice. Lysates were centrifuged (12 000 rpm, 12 min) and supernatants were incubated with 5 ug primary antibodies, which were coupled to magnetic beads according to the manufacturer's protocol (Invitrogen) or beads only for 2 h at 4°C, pulled down, washed 3× for 10 min with 800 μl lysis buffer at 4°C and eluted with SDS-PAGE sample buffer by boiling (5 min, 95°C).
For immunoprecipitation, cells were trypsinized, washed in cold 1× PBS and centrifuged (1200rpm, 5 min). Pellets were lysed in 5 volumes lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, 100 U RNase inhibitor, 1× protease/phosphatase inhibitor cocktails, Roche) for 20 min on ice. Lysates were centrifuged (12 000 rpm, 12 min) and supernatants were incubated with 5 ug primary antibodies, which were coupled to magnetic beads according to the manufacturer's protocol (Invitrogen) or beads only for 2 h at 4°C, pulled down, washed 3× for 10 min with 800 μl lysis buffer at 4°C and eluted with SDS-PAGE sample buffer by boiling (5 min, 95°C).
10
Click Chemistry for Protein Labeling
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Immunopurified proteins were clicked on beads by a 30-min incubation at 20 °C with vigorous intermittent shaking in 200 µL of IPD buffer containing 4 mM CuSO4, 5 µM azido-AlexaFluor647 and 10 mM sodium ascorbate. The beads were washed with IPD buffer and resuspended in 20 µL SDS-Lysis Buffer to which 20 µL of SDS Loading buffer was added. Beads were incubated 5 min at 95 °C in this solution and 20 µL of supernatant was analyzed on gradient gels (BioRad 4–12% TGX pre-cast gels). The modified proteins were detected with an infrared imager (Odyssey, LI-COR Bioscience) after transfer onto a nitrocellulose membrane (Protran, 45 µm pores, GE Healthcare) and total proteins on the membrane were stained with Ponceau S.
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