Human WT pED-FLAG-LMAN1, pED-FLAG-LMAN1 DCRD (R44-E296), pED-FLAG-LMAN1 DHelix (G271-N457), and pED-FLAG-LMAN1 N156A were reported previously.63 (link) Transient transfection of HEK293T cells or NIH/3T3 was performed using calcium phosphate transfection. Cell lysates for western blot were obtained using cell lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM β-glycerophosphate, 1 mM PMSF, 1 mM NaVO4, 10 mM calyculin A, protease inhibitor cocktail]. Cell lysates were ran on 10% percent bis/acrylamide gels using a Mini-Protean Tetra cell apparatus and transferred to nitrocellulose membranes using a Criterion blotter (all from Bio-Rad; Hercules, California) (Cat# 1658000, Cat# 1620112, Cat# 1704071). LMAN1 was detected using an α-LMAN1 antibody (Cell Signaling; Danvers, MA) (Cat# 13947) or using an α-FLAG antibody (MilliporeSigma; Burlington, MA) (Cat# F1804). Total protein was detected using stain free gels (Bio-Rad; Hercules, California) (Cat# 1610182).
Criterion blotter
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Criterion Blotter is a laboratory equipment designed for the transfer of proteins, nucleic acids, or other biomolecules from polyacrylamide gels to a membrane support, such as nitrocellulose or PVDF. It provides a reliable and efficient method for the analysis and detection of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence western blotting kit, by Roche (6 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (5 mentions)
Criterion cell, by Bio-Rad (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Criterion tgx precast gel, by Bio-Rad (4 mentions)
P erk, by Cell Signaling Technology (3 mentions)
Goat anti rabbit igg hrp, by Roche (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Nitrocellulose membranes 0.45 μm, by Thermo Fisher Scientific (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Image studio software, by LI COR (2 mentions)
Eif2α, by Thermo Fisher Scientific (2 mentions)
Phospho perk thr980, by Cell Signaling Technology (2 mentions)
Phospho eif2α ser51, by Cell Signaling Technology (2 mentions)
Nupage 8 bis tris midi gel, by Thermo Fisher Scientific (2 mentions)
Myc tag, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (2 mentions)
69 protocols using criterion blotter
1
LMAN1 Protein Expression and Analysis
2
Western Blot Analysis of Nuclear Proteins
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Nuclear proteins were extracted using a nuclear protein extraction kit according to the manufacturer’s instructions (Beyotime Co., Nantong, China). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Beyotime). Aliquots of cell lysates containing 20 μg protein were separated in 10% polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore) using a Bio-Rad criterion blotter. Membranes were blocked in 5% non-fat dried milk in TBST at room temperature for 1 h and then incubated with either anti-phospho-CREB (1:3000 dilution; Millipore) or anti-CREB (1:2000 dilution; Millipore) at 4 °C overnight. The blots were then washed and incubated with horseradish peroxidase-labeled secondary antibodies (Sigma) at 37 °C for 1 h. Immunoreactive bands were detected with enhanced chemiluminescence Western blotting detection reagents (Beyotime). The blots were exposed to X-ray film for radiography of the bands and were digitally detected and measured using a LAS3000 Bioimage Analyzer (Fuji Photo Film, Tokyo, Japan).
3
Heme-Staining and Immunoblotting Analysis
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Unless otherwise noted, cells of the late exponential phase were harvested, washed with phosphate buffered saline (PBS), resuspended in the same buffer, and sonicated. Protein concentrations of the cell lysates was determined by the bicinchoninic acid assay (Pierce Chemical). For heme-staining, the cell lysates were separated on SDS-PAGE using 12% polyacrylamide gels and stained with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as described elsewhere43 (link). Immunoblotting analysis was performed essentially the same as previously described44 (link). Proteins separated on SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with rabbit polyclonal antibodies against NrfA. The goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics) in accordance with the manufacturer’s instructions. Images were visualized with a UVP imaging system.
4
Cell Lysis and Western Blotting Protocol
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Sample preparation and western blotting were performed as previously described [21 (link)]. Cells, including detached ones, were freshly lysed in cold caspase lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 1% NP-40) reconstituted with 0.25 mM AEBSF and Halt Protease and Phosphatase Inhibitor Cocktail (78440, Thermo Scientific). Cell debris was removed by high-speed centrifugation for 20 min at and 4 °C, and protein concentration was quantified with Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Briefly, 25 μg of protein were resolved on Bolt 4–12% Bis-Tris Plus Gels (NW04125BOX, Invitrogen) under reducing conditions according to manufacturer’s instructions. After transfer onto PVDF membranes (IPVH00010, Millipore) using Criterion blotter (1704070, Bio-Rad), blocked membranes were incubated overnight at 4 °C with the indicated primary antibodies, washed and stained with secondary antibodies for 1 h at RT. Images were detected with WesternBright Quantum HRP substrate (K-12042-D10, Advansta) on LAS-3000 Imager (Fujifilm) and processed using ImageJ software with brightness/contrast adjustment applied to an entirely digital image if necessary.
5
Western Blot Analysis of CTIF and LRRN4
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Protein extracts were prepared in RIPA cell lysis buffer and each protein sample (1 μg/μL, 10 μL) was electrophoresed through a precast gel (NuPAGE®Novex® 4–12% Bis-Tris Gel, 1.5 mm, 10 wells, Invitrogen™, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane by means of the semidry technique using the Criterion Blotter (Bio-Rad) at 100 V for 60 min, and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween-20. Membranes were then separately incubated overnight with primary antibodies (CTIF, LRRN4, 1 μg/μL, 1:500). After washing, membranes were incubated with HRP-conjugated secondary antibodies for one hour (1:10000). Proteins were detected with an enhanced chemiluminscent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software.
6
Detection of His6-tagged Proteins by Western Blotting
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Western blotting analysis was performed for detection of His6-tagged proteins as previously described (Dong et al., 2012 (link)). Cells entering the stationary phase were harvested by centrifugation, washed with PBS (pH 7.0), resuspended in the same buffer, and sonicated. The cell lysates containing the same amount of proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10%). Proteins were then electrophoretically transferred to polyvinylidene difluoride membranes according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with a rabbit polyclonal antibody against His6-tag. Goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics). Images were visualized with a UVP imaging system.
7
SDS-PAGE and Western Blot Protocol
8
Western Blot Analysis of Cellular Stress Pathways
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Total cell lysates were made from mouse pancreatic tissue using RIPA buffer (1% Nonidet P40, 0.5% sodium doxycholate, 0.1% SDS, 1× PBS [pH 8.0]) with 1× Protease Inhibitor cocktails and 1× Phosphatase Inhibitor cocktail 2 and 3 (Millipore-Sigma). Lysate proteins from tissues or MEF cells were denatured by boiling the lysates in 2× SDS sample buffer for 5 min prior to electrophoresis on NuPAGE 8% Bis-Tris Midi gel (Invitrogen). The separated proteins were transferred to nitrocellulose membranes (0.45 μm, Thermo Scientific, Waltham, MA, USA) in carbonate transfer buffer using wet transfer conditions (Criterion Blotter, Bio-Rad). Primary antibodies (diluted in 5% BSA-TBST) used include: phospho-PERK (Thr980) (#3179, Cell Signaling, Danvers, MA, USA), PERK (#3192, Cell Signaling), phospho-eIF2α (Ser51) (#9721, Cell Signaling), eIF2α (#AHO1182, Invitrogen), Myc Tag (#R950-25, Invitrogen), and actin (#A5060, Millipore-Sigma). Appropriate IRDye-conjugated secondary antibodies were used, and IR fluorescence was detected using the LI-COR Odyssey CLx Imaging System and quantified using the LI-COR Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA).
9
Western Blot Analysis of Protein Lysates
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Cells were lysed in RIPA buffer (Cell signaling technology, 9806) with protease/phosphatase inhibitor cocktail (Cell signaling technology, 5872) and agitated at 4 °C for 30 min, followed by centrifugation at 2000 g for 20 min. Supernatants were collected and protein concentration was calculated by the Pierce BCA Protein Assay Kit (ThermoFisher Cat # 23225) according to manufacturer’s protocol. Equal amounts of protein are diluted in SDS running buffer and reducing reagent (ThermoFisher #NP0002 and #NP0004) and subjected to electrophoresis and immunoblotting has been performed according to manufacturer’s protocol (BioRad MiniProtein Electrophoresis cell #1658004 and BioRAD Criterion Blotter #1704070). The immunoblot membranes were blocked in 5% milk in TBST 1 h at room temperature, washed (TBST, 10 min) and incubated with primary antibodies diluted in 5% milk or BSA (for phospho antibodies) in overnight at 4 °C followed by a rigorous wash (5 min x4). Secondary antibody of appropriate species was diluted in 5% milk or BSA in TBST at 1:5000 dilution and incubated at room temperature for 1 h. After washing, membranes were incubated with chemiluminescence substrate (ThermoFisher # 34580) and imaged using (BioRAD ChemiDoc). Antibodies are listed in Table S1 .
10
Western Blot Analysis of TTR and LRP1
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The presence of TTR in human serum after depletion, as well as LRP1 total levels in brains and livers was studied by western blot analysis. Protein extract denatured samples (30–50 μg) were separated in SDS-PAGE gels (10% for LRP1 and 15% for TTR). Proteins were then transferred to nitrocellulose membrane (WhatmanTM Ge healthcare – Protan BA 83), using a wet system (Bio-rad Criterion Blotter). The membranes were blocked 1 hour at RT with 5% powered skimmed milk in PBS containing 0,05% Tween-20 (PBS-T). After blocking, membranes were then incubated with primary antibodies in 3% powered skimmed milk/PBS-T against the proteins under study: rabbit anti TTR (DAKO, 1:2000), rabbit anti LRP1 (Abcam, 1:15000), mouse anti Tubulin (Sigma-Aldrich, 1:2000), mouse anti β-Actin (Sigma-Aldrich, 1:3000). Then washed membranes were incubated for 1 hour at RT with sheep anti-rabbit (The binding Site; 1:10000) or anti-mouse (The binding Site; 1:2500) immunoglobulins conjugated with horseradish peroxidase in 3% powered skimmed milk/PBS-T. The blots were developed using ClarityTM Western ECL substrate (Bio-rad) and proteins were detected and visualized using a chemiluminescence detection system (ChemiDoc, Bio-rad).
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