Mitochondria-enriched and cytosolic fractions were isolated from cortex, hippocampus, cerebellum and olfactory bulb of WT and Wdfy3+/lacZ mice as described before68 (link). Thirty-five µg of proteins were solubilized in SDS sample buffer (Life Technologies, Grand Island, NY) and loaded onto a 4–12% bis-tris gel (Life Technologies) as previously described68 (link). After transferring proteins with an iBlot apparatus (Life Technologies), membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20 °C and subsequently probed overnight at 4 °C with the following antibodies: anti-Lamp2 (Abcam, Cambridge, MA; 1:1,000 dilution), anti-LC3 (Novus Biologicals, Littleton, CO; 1:1,000 dilution), anti-Mfn2 (Proteintech, Rosemont, IL; 1:500 dilution), anti-MnSOD (Millipore, Billerica, MA; 1:1,000 dilution), anti Sqstm1 (Cell Signaling Technology, Danvers, MA; 1:500 dilution), anti-Park2 (Abcam; 1:500 dilution), and anti-Pink1 (Novus Biologicals; 1:1,000 dilution). As a loading control, we used anti-β-actin antibody (Sigma, St. Louis, MO; 1:20,000 dilution, 1 h at 20 °C). Secondary antibodies were from LI-COR (Lincoln, NE; 1:10,000 dilution). Membranes were visualized with the use of the Odyssey Infrared Imaging System (LI-COR) and densitometry analysis carried out with ether the Carestream or ImageJ softwares.
Iblot apparatus
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States
The IBlot apparatus is a laboratory equipment designed for the transfer and detection of proteins from polyacrylamide gels to membranes. It provides a controlled and efficient method for the blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris gel, by Thermo Fisher Scientific (4 mentions)
Qiashredder column, by Qiagen (3 mentions)
Sds sample buffer, by Bio-Rad (2 mentions)
Sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Anti mnsod, by Merck Group (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti lamp2, by Abcam (1 mentions)
Anti mfn2, by Proteintech (1 mentions)
Anti sqstm1, by Cell Signaling Technology (1 mentions)
Anti park2, by Abcam (1 mentions)
Anti pink1, by Novus Biologicals (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Quikchange method, by Agilent Technologies (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Odyssey image station, by LI COR (1 mentions)
23 protocols using iblot apparatus
1
Organelle Fractionation and Western Blotting
2
Transient Expression and Purification of T1r Proteins
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Transient expression of T1rLBD proteins in S2 cells was carried out by the calcium phosphate method as described previously22 (link), or by using FuGENE HD (Roche) with 1 μg each T1r2aLBD and T1r3LBD expression vectors for 1 × 106 cells according to the manufacturer's protocol. The cells were cultivated at 300 K for 4 days. Mutations were introduced using the QuikChange method (Agilent Technologies).
The cell culture supernatant was mixed with ANTI-FLAG M2 affinity gel (SIGMA) and rotated at 277 K. After washing with 20 mM Tris, pH 7.4, containing 150 mM NaCl, the proteins retained on the beads were eluted with 2 × SDS–PAGE sample buffer (100 mM Tris, 2% SDS, 10% (v/v) glycerol, 0.002% bromophenol blue, pH6.8), followed by heating at 368 K for 5 min. The eluents were divided into two equal parts, further incubated at 368 K for 5 min with or without 100 mM DTT, and subjected to SDS-PAGE followed by electrophoretic transfer onto membranes, using an iBlot apparatus (Life Technologies). T1rLBDs were detected using anti-DDDDK tag-HRP (1:2,000) (Cat. # PM020-7, Medical and Biological Laboratories) and Immobilon Western Chemiluminescent HRP Substrate (Millipore). The images were obtained using a ChemiDoc Imager (Bio-Rad). The uncropped original images of the blots are shown inSupplementary Fig. 9 .
The cell culture supernatant was mixed with ANTI-FLAG M2 affinity gel (SIGMA) and rotated at 277 K. After washing with 20 mM Tris, pH 7.4, containing 150 mM NaCl, the proteins retained on the beads were eluted with 2 × SDS–PAGE sample buffer (100 mM Tris, 2% SDS, 10% (v/v) glycerol, 0.002% bromophenol blue, pH6.8), followed by heating at 368 K for 5 min. The eluents were divided into two equal parts, further incubated at 368 K for 5 min with or without 100 mM DTT, and subjected to SDS-PAGE followed by electrophoretic transfer onto membranes, using an iBlot apparatus (Life Technologies). T1rLBDs were detected using anti-DDDDK tag-HRP (1:2,000) (Cat. # PM020-7, Medical and Biological Laboratories) and Immobilon Western Chemiluminescent HRP Substrate (Millipore). The images were obtained using a ChemiDoc Imager (Bio-Rad). The uncropped original images of the blots are shown in
3
Western Blot Analysis of Proteins
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Samples (purified protein or cell lysate) were resolved on a 12% SDS gel and transferred onto a nitrocellulose membrane using i-blot apparatus (Life Technologies). The membrane was washed with 1× PBS, dried, and blocked overnight with 5% blotting-grade blocker in PBST (PBS with 0.05% Tween-20). The block was removed and the membrane was incubated with antibodies (c-Myc [9E10] HRP, mouse monoclonal IgG1, His-probe [H-3] HRP, or mouse monoclonal IgG1, each at 1:1000 dilution). After 30-min incubation on a rocker, the membrane was washed thrice with PBST and protein bands were detected with a chemiluminescent HRP substrate.
4
Serotyping of Klebsiella pneumoniae Isolates
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The serotypes of the KP clinical isolates were determined by western blot analysis with serotype-specific monoclonal antibodies or mouse polysera. Briefly, purified LPS or bacterial lysates were subjected to SDS-PAGE. Separated proteins and LPS were transferred from gels to nitrocellulose membranes with an iBlot apparatus (Life Technology). Membranes were then blocked with Casein or Odyssey (Li-cor) blocking buffer before being probed with anti-O1, O1/O2, O3, O4, or O5 LPS monoclonal antibodies or anti-O7 and anti-O12 mouse polysera. After repeated washes with PBS-T, blots were incubated with IRDye680 or 800 fluorescent secondary antibodies (Li-cor) and visualized with an Odyssey Image Station (Li-cor). In some circumstances, bacterial lysates were treated with 0.4 mg ml−1 Proteinase K (ThermoScientific) to remove protein components before the western blot analysis. CRE isolates were defined as resistant to carbapenams (doripenam, imipenam and/or meropenam MIC > = 4 μg ml−1), ESBL isolates defined as resistant to cephalosporins (ceftazidime MIC > = 16 μg ml−1) but sensitive to carpapenams (MIC < 4 μg ml−1) and susceptible isolates defined by ceftazidime MIC < 16 μg ml−1 and carbapenam MIC < 4 μg ml−1.
5
Western Blot Analysis of Neuronal Proteins
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Tissues and
MEFs were harvested and lysed in RIPA buffer (Pierce) containing complete
mini EDTA-free protease inhibitor cocktail (Roche) plus phosphatase
inhibitor cocktail 2 and 3 (Sigma-Aldrich). Total lysates were resolved
by SDS-PAGE and transferred to nitrocellulose membranes using the
iBlot apparatus (Invitrogen). Immunoblot analysis was performed per
standard procedures using commercially available antibodies (Atp5a,
Hspa8, Pacsin1, Uchl1, Mog, ubiquitin (Santa Cruz Biotechnologies);
Mapt, Ppia, Sirt2 (Sigma); Sod1 (Millipore); Dnm1 (Cell Applications);
and Snap25, Stmn1, Parp (Cell Signaling). All of the primary antibodies
were used at dilutions of 1:1000. Horseradish-peroxidase-conjugated
secondary antibodies were used at dilutions of 1:2000 (Invitrogen).
Proteins were visualized using ECL (Cell Signaling Technology) and
autoradiography. Densitometry of immunoreactive bands was performed
using ImageJ software. p values between two comparisons
were calculated using a two-tailed t test from the
average raw values of pixel densities of bands in triplicate where
indicated. Pearson correlation coefficients were calculated from the
average of the raw values of pixel densities of bands in triplicate
using the ratio of various comparisons for the biological and technical
replicates, where indicated.
MEFs were harvested and lysed in RIPA buffer (Pierce) containing complete
mini EDTA-free protease inhibitor cocktail (Roche) plus phosphatase
inhibitor cocktail 2 and 3 (Sigma-Aldrich). Total lysates were resolved
by SDS-PAGE and transferred to nitrocellulose membranes using the
iBlot apparatus (Invitrogen). Immunoblot analysis was performed per
standard procedures using commercially available antibodies (Atp5a,
Hspa8, Pacsin1, Uchl1, Mog, ubiquitin (Santa Cruz Biotechnologies);
Mapt, Ppia, Sirt2 (Sigma); Sod1 (Millipore); Dnm1 (Cell Applications);
and Snap25, Stmn1, Parp (Cell Signaling). All of the primary antibodies
were used at dilutions of 1:1000. Horseradish-peroxidase-conjugated
secondary antibodies were used at dilutions of 1:2000 (Invitrogen).
Proteins were visualized using ECL (Cell Signaling Technology) and
autoradiography. Densitometry of immunoreactive bands was performed
using ImageJ software. p values between two comparisons
were calculated using a two-tailed t test from the
average raw values of pixel densities of bands in triplicate where
indicated. Pearson correlation coefficients were calculated from the
average of the raw values of pixel densities of bands in triplicate
using the ratio of various comparisons for the biological and technical
replicates, where indicated.
6
Western Blot Analysis of HIF-1α and ISCU
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Cells were lysed and cytoplasmic and nuclear extracts were prepared using the NE-PER extraction kit with 1X halt-protease inhibitors cocktail (both Pierce, Waltham, MA) and EDTA. Protein extracts were stored at -80°C. 20μg of protein were mixed with warm 2X lithium dodecyl sulfate (LDS) such that the LDS to sample ratio was 1:4 (v/v). Sample were denatured for 5 minutes at 95°C and subjected to SDS-PAGE (4–12% NuPAGE Bis-Tris) with 1X MOPS running buffer diluted with dH2O (all Invitrogen). The proteins were transferred onto a nitrocellulose membrane by an iBlot apparatus for 8 minutes (all Invitrogen). The membrane was blocked with 5% w/v nonfat dry milk in 1X TBST (10nM Tris-HCl, pH 8.0, 150nM NaCl, and 0.05% Tween 20). The blot was incubated with mouse antibodies to HIF-1α (1:500, BD Biosciences, San Jose, CA) or ISCU (1:800, ab180532, Abcam) overnight and goat anti-mouse IRDye 800CW antibody (Licor Biosciences, Lincoln, NE) at a 1:10,000 dilution for 30 minutes; being washed twice with washing buffer in between incubations. Scanning was performed using the Li-Cor Odyssey CLx imaging system coupled with the Image Studio software. Images were later adjusted for contrast and intensity using PowerPoint 2011 version 14.6.3 for Macintosh (Microsoft Co, Redmond, WA).
7
Western Blot Protein Analysis
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Cells were lysed using RIPA cell lysis reagent (Thermo Fisher Scientific) and protease inhibitors cocktail (Sigma-Aldrich). Equal amounts of proteins were subjected to SDS-PAGE on 4%–12% Bis-Tris gel. Proteins were transferred onto a nitrocellulose membrane (Life Technologies) using the iBlot apparatus (P3 program, 7 min transfer, Invitrogen). The membrane was exposed to Ponceau red staining (Sigma-Aldrich), washed, incubated in 5% non-fat dried milk in TBS containing 0.05% Tween-20 buffer for 1 hr at room temperature (RT), and subsequently incubated overnight at 4°C with primary antibody against PAXX protein (ab126353, 1:1000 dilution, Abcam) and γ-Tubulin protein (T6557, 1:20,000 dilution, Sigma-Aldrich). The membrane was then washed three times with TBS-Tween before incubation for 1 hr at RT with HRP-conjugated antibodies (7,074 or 7,076, 1:20,000 dilution, Cell Signaling Technology). Immune complexes were detected with WesternBright Sirius substrate (Advansta).
8
Protein Extraction and Western Blot Analysis
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Cells were lysed in PBS + 1% SDS and passed through a Qiashredder column (Qiagen) to disrupt DNA. For human muscle, protein extracts were obtained from 200–300 sections from each biopsie and lysed in RIPA buffer.
Protein concentration was measured with a BCA kit according to manufacturer instructions (Pierce). Equal amount of sample were boiled in 30 µl sample buffer and were loaded on 4–12% precast Bis-Tris gel (Invitrogen) and transferred into nitrocellulose membrane using the iBlot apparatus (Invitrogen). Membranes were blocked with blocking buffer (5% non-fat dry milk, 0.1% Tween in TBS). Primary antibodies were incubated overnight in blocking buffer at 4°C. After three washes with TBS-Tween 0.1%, membranes were incubated with HRP-conjugated secondary antibodies (1 h at room temperature). Proteins were visualized using ECL reagent (Pierce).
Protein concentration was measured with a BCA kit according to manufacturer instructions (Pierce). Equal amount of sample were boiled in 30 µl sample buffer and were loaded on 4–12% precast Bis-Tris gel (Invitrogen) and transferred into nitrocellulose membrane using the iBlot apparatus (Invitrogen). Membranes were blocked with blocking buffer (5% non-fat dry milk, 0.1% Tween in TBS). Primary antibodies were incubated overnight in blocking buffer at 4°C. After three washes with TBS-Tween 0.1%, membranes were incubated with HRP-conjugated secondary antibodies (1 h at room temperature). Proteins were visualized using ECL reagent (Pierce).
9
Western Blotting for Protein Detection
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Cells were lysed in PBS + 1%SDS and passed through a Qiashredder column (Qiagen) to disrupt DNA. Protein concentration was measured with a BCA kit according to manufacturer instructions (Pierce). Equal amount of sample were boiled in 30ul sample buffer and were loaded on 4-12% pre-cast Bis-Tris gel (Invitrogen) and transferred into nitrocellulose membrane using the iBlot apparatus (Invitrogen). Membranes were blocked with blocking buffer (5% Non Fat Dry Milk, 0.1% Tween in TBS). Primary antibodies were incubated overnight in blocking buffer at 4°C. After three washes with TBS-Tween 0.1%, membranes were incubated with HRP-conjugated secondary antibodies (1 hour at room temperature). Proteins were visualized using ECL reagent (Pierce). All western blots were performed 3 times.
10
Phosphorylation Analysis of Smc4 and Ycg1
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Eight percent Phos-tag acrylamide gels (Wako Chemicals USA) were used to resolve Smc4-NT phosphorylated species (Kinoshita et al. 2006 (link)), whereas gels containing 7.5% Next gel acrylamide (Amresco) were used to monitor Smc4 abundance and Ycg1 phosphorylation (St-Pierre et al. 2009 (link)). All gels were transferred using the iBlot apparatus (Invitrogen). Antibodies and conditions used for immunoblotting are listed in the Supplemental Material.
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