Western blotting was performed using the Bio-Rad wet-transfer method as described previously (Xie et al. 2022 (link)). Briefly, for western blotting of whole worm extracts, 10–14 µl of extracts were loaded onto each well of a 7.5% SDS-PAGE gel. The gels were run until proper band separation was achieved and the protein was transferred onto a 0.2 µm nitrocellulose membrane using the wet-transfer method (Bio-Rad Laboratories). The membranes were blocked with Odyssey Blocking Buffer (LiCOR Biosciences, Inc.), probed with anti-HA (Cell Signaling, catalog C29F4 Rabbit mAb #3724) and anti-tubulin (Santa Cruz Biotechnology, Inc., catalog sc-32293) antibodies overnight at 4°C. The membranes were washed thrice with 1× TBST (0.1% w/v Tween-20, 50 mM Tris–Cl, pH 7.6, 150 mM NaCl) and incubated with IRDye 800CW Donkey anti-Rabbit IgG secondary antibody (LiCOR Biosciences, Inc. catalog # 926-32213) and IRDye 680RD Goat anti-Mouse IgG secondary antibody (LiCOR Biosciences, Inc. catalog # 926-68070) for 1 h at room temperature. The membranes were washed again thrice with 1XTBST and imaged using the LI-COR Odyssey CLx imager (LI-COR Biosciences, Inc.). The band intensities were adjusted equally for all the gel lanes for better data visualization and presentation. Supplementary Table 3 lists all the antibodies that have been used in this study.
Wet transfer method
Manufactured by Bio-Rad
Sourced in United States
The wet transfer method is a laboratory technique used to transfer proteins from a gel to a membrane, such as nitrocellulose or PVDF, for further analysis. The process involves the use of an electrical current to drive the transfer of proteins from the gel to the membrane, allowing for the detection and analysis of specific proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Revert stain, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
C digit blot scanner, by LI COR (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
N ter, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Tbs 0.1 tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab150377, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti mouse or anti rabbit secondary antibodies, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab8245, by Abcam (1 mentions)
Sds page, by Beyotime (1 mentions)
Two color infrared laser imaging system, by LI COR (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
11 protocols using wet transfer method
1
Western Blotting of Worm Extracts
2
Western blot analysis of Dpy19L1
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COS-7 cells were transfected with a pCAG-Dpy19L1 plasmid. Forty-eight h later, they were lyzed with Cell-LyEX MP (TOYO INK). Cell lysates were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore) by wet transfer method (Bio-Rad). Following blocking with 5% nonfat dry milk, the membrane was incubated with rabbit anti-Dpy19L1 (N-ter, 1:500, Abcam), rabbit anti-Dpy19L1 (C-ter, 1:500, Abgent), or mouse anti-α-Tubulin (1:4000, Sigma) overnight at 4°C and then reacted with the secondary antibody conjugated to horseradish peroxidase (1: 10000, MBL). Signals were developed using Immunostar LD (Wako) and scanned with a C-DiGit Blot Scanner (LI-COR).
3
Western Blot Analysis of p70 S6 Kinase
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Brains were removed and homogenized in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% w/v Triton X-100) supplemented with Roche Complete protease inhibitor cocktail, using a motorised pellet mixer with autoclaved polypropylene pestles. Twenty μg of total protein homogenates were resolved in 10% SDS-PAGE gels, transferred to PVDF membranes using a wet transfer method (BioRad) and incubated for 1 h with anti-p70 S6 Kinase antibody (49D7) (Rabbit mAb, 1:3000, #2708 S; Cell Signalling) in blocking solution (5% milk in TBS-Tween 0.1%). Tubulin was used as loading control (1:10,000, #T5293 Sigma). Detection was performed using enhanced chemiluminescence (Luminata Crescento, Millipore) and film exposure (Amersham Biosciences).
4
Protein Extraction and Western Blotting
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Cells were lysed with SDS lysis buffer (2% SDS, 10% Glycerol, 50mM Tris pH 6.8) and boiled at 95°C for 30min. Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFischer) following manufacturer’s recommendations. Samples were ran in NuPAGETM 10% Bis-Tris Midi electrophoresis gels (Invitrogen) using NuPAGETM MES SDS running buffer (ThermoFischer Scientific). Proteins were subsequently transferred to an Immobilon-P polyvinylidene Difluoride (PVDF) membrane (Merck Millipore) using the wet-transfer method (Bio-Rad) in a transfer buffer consisting of 20% (v/v) methanol and 10% protein electrophoresis buffer (24.77 mM Tris and 0.192 M glycine).
For western blotting membranes were blocked in a solution of 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) dissolved in TBS-0.1% Tween 20 (Sigma-Aldrich) then incubated at 4°C overnight with the primary antibodies diluted in 5% (w/v) BSA/TBS-T. The membranes where then washed and incubated with anti-mouse and/or anti-rabbit secondary antibodies (LI-COR Biosciences) for 2h at RT diluted in 5% (w/v) BSA/TBS-T then washed and imaged using the LI-COR Odyssey (LI-COR Biosciences). Images were analyzed using Image Studio Lite software (LI-COR Biosciences).
For western blotting membranes were blocked in a solution of 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) dissolved in TBS-0.1% Tween 20 (Sigma-Aldrich) then incubated at 4°C overnight with the primary antibodies diluted in 5% (w/v) BSA/TBS-T. The membranes where then washed and incubated with anti-mouse and/or anti-rabbit secondary antibodies (LI-COR Biosciences) for 2h at RT diluted in 5% (w/v) BSA/TBS-T then washed and imaged using the LI-COR Odyssey (LI-COR Biosciences). Images were analyzed using Image Studio Lite software (LI-COR Biosciences).
5
Quantitative Protein Analysis by Western Blot
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The protein in each sample was extracted through tissue homogenation. Similar protein concentrations were loaded into the gel, separated by SDS-PAGE gels electrophoresis (8–12% SDS–PAGE gels), and transferred to a nitrocellulose membrane using the wet transfer method (Bio-Rad, Hercules, CA, USA). The membranes were blocked with TBST (0.05% Tween-20 in TBS) containing 5% skim milk and then incubated overnight with PKM2 (ab150377, Abcam Inc., Cambridge, CA, USA) (1:1000) and GAPDH(ab8245, Abcam Inc., Cambridge, CA, USA) (1:5000) antibodies at 4 °C overnight. Next, the membranes were washed three times in TBST, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies(Bio-Rad, Hercules, CA, USA) (1:5000) for 60 minutes at room temperature. Western Blotting Lightning Reagent (203-15291; PerkinElmer) was used to detect the results.
6
SDS-PAGE and Western Blot Analysis
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The denatured proteins were separated with 12% SDS‐PAGE (Beyotime, China). The electrophoretic conditions were 100V for 20 min and 120 V for 60 min. The protein was transferred to PDVF membrane by wet transfer method (BioRad). The membrane was incubated with the first antibody, and then secondary antibody (ZenBio) was used for protein labeling. The protein bands were developed using the color‐substrate solution (Beyotime) and photographed in a two‐color infrared laser imaging system (Li‐cor).
7
Protein Extraction and Analysis Protocol
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For total protein extraction, after being treated with different concentration of chitopentaose, HepG2 cells were lysed with a RIPA (Thermo, Waltham, MA) and 1 mM PMSF (Beyotime, Shanghai, China) cocktail using an ultrasonic cell disruptor. Separation of cytosolic protein and mitochondrial protein was performed using a cytosol-mitochondrial isolation kit (Beyotime, Shanghai, China). Protein concentration was analyzed using a BCA kit (Beyotime, China). An equal amount of protein was electrophoresed by 12% or 15% sodium laurylsulfonate (SDS) gel and transferred to polyvinylidene fluoride membrane by the wet transfer method (Bio-rad, Hercules, CA). The membrane was blocked with TBST (20 mM Tris-base, 150 mM NaCl, 0.1% Tween-20) containing 5% (m/v) non-fat milk powder and incubated with primary antibody at proper dilutions overnight at 4 °C. After being washed 3 times with TBST, the membrane was incubated with secondary antibody (1:3000) for 1 h at room temperature. The immunoblot on the membrane was developed with an enhanced chemiluminescence system. β-actin was used as an internal reference. The relative protein concentration was expressed as folds of the control group. The intensity of the immunoblot was quantified with ImageJ software.
8
Western Blot Analysis of Hippocampal Proteins
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Hippocampal lysates supplemented were denatured at 95°C using the 2-Mercaptoethanol based loading buffer. Proteins were separated using the SDS-polyacrylamide gel electrophoresis and western blot procedure. Custom made 8-10% gels were used to run the samples for 1.5 hours at 110 voltage. The proteins were then transferred to a precut 0.2 µm nitrocellulose membrane (Cat: 1620146; Biorad, USA) using a wet transfer method (Biorad). Later the membranes were incubated in a blocking solution containing 0.5% bovine serum albumin (Art. No. 8076.4, Roth, Germany), 1xTBS, and 0.1%Tween-20 for 30 minutes at room temperature on a rotating shaker. The membranes were then incubated with primary antibodies overnight at 4°C with gentle agitation. On the second day, the membranes were rinsed with TBS-Tween solution 3 x 5 minutes, followed by a 1-hour incubation with the uorescently conjugated secondary antibodies, rinsed again and air-dried covered by aluminum foil. The proteins were detected using the Omega Lum (Labgene, CH). The optical density of the protein bands was determined using Image J software and normalized to the Beta-actin control. For an accurate total protein quanti cation REVERT stain (LI-COR Biosciences-GmbH, Germany) was also employed.
9
Western Blot Analysis of Hippocampal Proteins
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Hippocampal lysates were denatured at 95°C using the 2-Mercaptoethanol based loading buffer. Proteins were separated using the SDS-polyacrylamide gel electrophoresis and western blot procedure. Custom made 8-10% gels were used to run the samples for 1.5 hours at 110 voltage. The proteins were then transferred to a precut 0.2 µm nitrocellulose membrane (Cat: 1620146; Biorad, USA) using a wet transfer method (Biorad). Later the membranes were incubated in a blocking solution containing 0.5% bovine serum albumin (Art. No. 8076.4, Roth, Germany), 1xTBS, and 0.1%Tween-20 for 30 minutes at room temperature on a rotating shaker. The membranes were then incubated with primary antibodies overnight at 4°C with gentle agitation. On the second day, the membranes were rinsed with TBS-Tween solution 3 x 5 minutes, followed by a 1-hour incubation with the uorescently conjugated secondary antibodies, rinsed again and air-dried covered by aluminum foil. The proteins were detected using the Omega Lum (Labgene, CH). The optical density of the protein bands was determined using Image J software and normalized to the Beta-actin control. For an accurate total protein quanti cation REVERT stain (LI-COR Biosciences-GmbH, Germany) was also employed.
10
Western Blot Analysis of Hippocampal Proteins
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Hippocampal lysates supplemented were denatured at 95°C using the 2-Mercaptoethanol based loading buffer. Proteins were separated using the SDS-polyacrylamide gel electrophoresis and western blot procedure. Custom made 8-10% gels were used to run the samples for 1.5 hours at 110 voltage. The proteins were then transferred to a precut 0.2 µm nitrocellulose membrane (Cat: 1620146; Biorad, USA) using a wet transfer method (Biorad). Later the membranes were incubated in a blocking solution containing 0.5% bovine serum albumin (Art. No. 8076.4, Roth, Germany) , 1xTBS, and 0.1%Tween-20 for 30 minutes at room temperature on a rotating shaker. The membranes were then incubated with primary antibodies overnight at 4°C with gentle agitation. On the second day, the membranes were rinsed with TBS-Tween solution 3 x 5 minutes, followed by a 1-hour incubation with the fluorescently conjugated secondary antibodies, rinsed again and air-dried covered by aluminum foil. The proteins were detected using the Omega Lum (Labgene, CH). The optical density of the protein bands was determined using Image J software and normalized to the Beta-actin control. For an accurate total protein quantification REVERT stain (LI-COR Biosciences-GmbH, Germany) was also employed.
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