Surface biotinylation samples were heated to 50 °C for 10 min before separation using 4% to 20% polyacrylamide gradient gels (Bio-Rad, Hercules, CA, USA). After separation, proteins were transferred to nitrocellulose membranes using Invitrogen's Power Blotter System. Blots were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature while rocking. After blocking, blots were incubated overnight at 4 °C with a mouse antibody against the α subunit of Na+/K+-ATPase (Abcam-ab7671; 1:2000) and with a mouse antibody against the His tag (Tetra·His Antibody, QIAGEN catalog no. 34670; 1:2000) in blocking solution on a rocker. The next day blots were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 and TBS before incubation with a horseradish peroxidase–conjugated goat antimouse secondary antibody at 1:10,000 (Thermo Fisher Scientific, catalog no. 31430) in 2.5% milk in TBS. After 1 h, blots were washed with TBS and incubated with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). Blots were visualized using a LI-COR Odyssey Fc (LI-COR, Lincoln, NE, USA), and bands were quantified using their Image Studio Lite Quantification Software.
Power blotter system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Power Blotter System is a versatile lab equipment designed for the transfer of proteins from polyacrylamide gels to membranes. It provides consistent and reliable performance for Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Calnexin, by Cell Signaling Technology (2 mentions)
Ibright imaging system, by Thermo Fisher Scientific (2 mentions)
Pierce rapid gold bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tetra his antibody, by Qiagen (1 mentions)
Image studio lite quantification software, by LI COR (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Horseradish peroxidase conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ab9106, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
14 protocols using power blotter system
1
Quantifying Membrane Protein Expression
2
Western Blot Analysis of Protein Targets
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Cells or tissue were lysed in RIPA buffer with 1:100 Halt protease inhibitor cocktail (both from Thermo Fisher Scientific). Total protein was quantified with bicinchoninic acid (BCA) protein assay (Pierce Biotechnology) to equalize the protein loaded among samples. For SDS-PAGE, samples were denatured and loaded on a NuPAGE 4–12% Bis-Tri gel in MOPS (Invitrogen). Protein was transferred to nitrocellulose membranes using Power Blotter System (Invitrogen). Blots were blocked in 5% bovine serum albumin (BSA) for at least 1 h and incubated with the primary antibody diluted 1:1000 in TBS 0.1% Tween (TBS-T) at 4 °C O/N. After washing 3 × in TBS-T blots were incubated with the HRP-linked secondary antibody diluted 1:10,000 in TBS-T, for at 1 h at RT. Blots were then washed 3 × in TBS-T and the signal developed using Immobilon Western (Millipore). Images were taken with Bio-Rad Chemidoc imager (BioRad). For all experiments, GAPDH was used as loading control. The following antibodies were used: GAPDH (Cell Signaling Technology), REST (Millipore), CTDSP1 (Invitrogen).
3
Protein Extraction and Western Blotting
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Cells corresponding to 2 units of OD600 were subject to protein extraction by TCA method. To the cell pellet 200 µl of 20% TCA was added and lysed by vortex at high speed for 5 min. The lysate was then collected onto a fresh tube and the glass beads were washed twice with 150 µl of 5% TCA. The washed elutes were added to the previous lysate and spun at 13000 rpm for 10 min. To the pellet 200 µl of Laemmli buffer was added and pH was adjusted using 2 M Tris.
The dissolved protein pellet was then denatured by boiling at 95°C for 5 min. SDS-PAGE was performed followed by a semi-dry method of transfer (Power Blotter system/Invitrogen) to PVDF membrane for western blotting. The blots were then probed with primary TAP-tag antibody (Genescript A01435, 1:5000) and HRP conjugated secondary anti-rabbit (Abcam ab97051, 1:10,000). For probing Rtt103 × 13myc, rabbit Myc (Abcam Ab9106 1:1000) primary was used. The signal was detected by ECL reagent (BioRad) and imaged in ChemiDoc Imaging system by BioRad.
The dissolved protein pellet was then denatured by boiling at 95°C for 5 min. SDS-PAGE was performed followed by a semi-dry method of transfer (Power Blotter system/Invitrogen) to PVDF membrane for western blotting. The blots were then probed with primary TAP-tag antibody (Genescript A01435, 1:5000) and HRP conjugated secondary anti-rabbit (Abcam ab97051, 1:10,000). For probing Rtt103 × 13myc, rabbit Myc (Abcam Ab9106 1:1000) primary was used. The signal was detected by ECL reagent (BioRad) and imaged in ChemiDoc Imaging system by BioRad.
4
Quantifying Smad2/3 Phosphorylation in Fibroblasts
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To assess the level of Smad2/3 phosphorylation, fibroblasts were lysed using RIPA buffer (1% NP40, 10 mM Tris-HCl pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 2 mM sodium orthovanadate, 10 mM sodium fluoride) supplemented with protease and phosphatase inhibitors (Sigma). Samples were denatured by boiling for 5 min at 95° degrees in Laemmli buffer and separated by SDS-Page using a 10% bis-tris acrylamide gel. Proteins were blotted on a nitrocellulose membrane using the semi-dry Power Blotter system (Invitrogen). Membranes were blocked in 5% BSA (for pSMAD2/3) or 5% skimmed milk (for total SMAD2/3) in TBS 0.1% Tween20 (TBST) for 30 min at room temperature, then incubated overnight at 4 °C with rabbit anti-Smad2/3 (Cell Signaling 8685) or rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling 8828), diluted 1:1000 in their blocking solution. After five washes with TBST, membranes were incubated with goat anti-rabbit HRP-conjugated antibody (DAKO, p0448), diluted in their blocking solution 1:2000 for 1 h at room temperature. After five washes with TBST, membranes were incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermoscientific) and chemiluminescence was detected using Chemidoc Touch imaging system (Promega).
5
Western Blot Analysis of Cell Lysates
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Total cell lysates were prepared using lysis buffer (Biosource International) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726, Sigma Aldrich), Phosphatase Inhibitor Cocktail 3 (P044, Sigma Aldrich), Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF serine protease inhibitor (Sigma-Aldrich), and 10 µg of proteins were separated by SDS-PAGE using Bolt 4–12% Bis–Tris Plus gels (Invitrogen). Gels were transferred onto nitrocellulose membranes using Power Blotter Select Transfer Stacks and the Power Blotter System (Invitrogen). Membranes were blocked with I-Block reagent (ThermoFisher) and incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-calnexin (sc-23954, Santa Cruz); mouse monoclonal anti-CD63 (ab213090, abcam); mouse monoclonal anti-CD81 (ab59477, abcam); mouse monoclonal anti-GAPDH (GTX627408); mouse monoclonal anti-Cytochrome C (SC-13156); mouse monoclonal anti-HSP70/HSC70 (W27, SC-24). Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody, Goat anti-Mouse (G-21040). Images were acquired using Westar Hypernova ECL substrate (Cyanagen) and iBright Western Blot Imaging Systems (Invitrogen).
6
Quantification of Fecal IgA Coating
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The samples containing fecal bacteria were prepared as described above. The bacterial pellet was lysed with 15 µl RIPA lysis buffer. Mouse serum IgA (Bethyl) was also lysed and used as
standard for the quantification of IgA. The bacterial lysate was mixed with 4 × Laemmli buffer and separated on 10% SDS-PAGE. The separated proteins were transferred onto a PVDF membrane
using the Power Blotter System (Thermo Fisher Scientific). After blocking with 1% skim milk buffer, the membrane was stained with anti-mouse IgA antibody (Bethyl) and HRP-conjugated
anti-goat IgG antibody (R&D Systems) as primary and secondary antibodies, respectively, and visualized using Chemi-Lumi One Super substrates (Nacalai). Imaging and quantification of IgA
coating fecal bacteria were performed using a ChemiDoc XRS+ system (Bio-Rad). The amount of IgA coating fecal bacteria was quantified by measuring the band intensity and comparing it to
reference bands of serum IgA.
standard for the quantification of IgA. The bacterial lysate was mixed with 4 × Laemmli buffer and separated on 10% SDS-PAGE. The separated proteins were transferred onto a PVDF membrane
using the Power Blotter System (Thermo Fisher Scientific). After blocking with 1% skim milk buffer, the membrane was stained with anti-mouse IgA antibody (Bethyl) and HRP-conjugated
anti-goat IgG antibody (R&D Systems) as primary and secondary antibodies, respectively, and visualized using Chemi-Lumi One Super substrates (Nacalai). Imaging and quantification of IgA
coating fecal bacteria were performed using a ChemiDoc XRS+ system (Bio-Rad). The amount of IgA coating fecal bacteria was quantified by measuring the band intensity and comparing it to
reference bands of serum IgA.
7
Epidermal Differentiation Protein Analysis
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After treatment, the RHE models were washed with cold PBS and harvested with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). A bicinchoninic Protein Assay kit (Thermo Fisher Scientific) was used to measure the protein concentrations. Then, 20 μg proteins were boiled and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gels were transferred onto a polyvinylidene fluoride (PVDF) membrane by using a Power Blotter System (Thermo Fisher Scientific). Following 5% skimmed-milk blocking, the membranes were incubated with anti-FLG (1:2000; Thermo Fisher Scientific), anti-IVL (1:2000, Novus Biological, Bio-Techne, Minneapolis, MN, USA), and anti-GAPDH (1:5000; Invitrogen, Thermo Fisher Scientific) antibodies overnight at 4 °C. The membranes were washed and incubated with a secondary antibody (1:20,000) (Invitrogen, Thermo Fisher Scientific) conjugated with horseradish peroxidase (HRP) in 0.5% PBST for 1 h. Protein expression was detected using an iBright FL1000 image system (Thermo Fisher Scientific) and quantified using iBright analysis software 3.0.0 (Thermo Fisher Scientific).
8
Exosomal Protein Immunoblotting Protocol
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Immunoblotting was performed as previously described (Tran et al., 2023 (link)). Briefly, exosome samples were lysed in the radioimmunoprecipitation assay (RIPA) lysis buffer and the total protein content was determined using the Pierce rapid gold BCA protein assay kit (Thermo Fisher, USA). Samples with equal protein loading were separated by SDS‐PAGE and then transferred semidry to a PVDF membrane using the Power Blotter system (Thermo Fisher, USA). The membranes were then blocked with 5% (w/v) bovine serum albumin (BSA) in tris‐buffered saline with the addition of 0.1% Tween‐20 (TBST) for 1 h before incubation with primary antibodies in the blocking buffer at 4°C overnight. The primary antibodies used were CD81 (#sc‐166029, Santa Cruz Biotechnology, CA, USA), CD9 (#ab236630, Abcam, Cambridge, UK), TSG101 (#sc‐7964, Santa Cruz Biotechnology), β‐actin (#sc‐47778, Santa Cruz Biotechnology), Calnexin (#2679, Cell Signaling), β‐Tubulin (#2128, Cell Signaling). After washing with TBST, the membranes were incubated with secondary antibody at room temperature for 1 h. Immunoblots were captured and quantified using the iBright imaging system (Thermo Fisher Scientific).
9
Quantifying Sumoylation Using Western Blot
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Protein samples were analyzed by standard polyacrylamide gel electrophoresis (PAGE) techniques. For preferential detection of high-molecular weight SUMO conjugates, PAGE was performed with lysates followed by wet transfer (100 V for 1 h) in a buffer consisting of 0.1% SDS, 10% methanol, 50 mM Tris-HCl, and 380 mM glycine. For preferential detection of free SUMO and lower molecular-weight SUMO conjugates, TCA extracts were analyzed by PAGE, followed by semi-dry transfer using a Power Blotter system (Thermo Fisher). Chemiluminescence-based imaging was performed using the MicroChemi imager (DNR). For quantification of signals, TIFF-format images were analyzed using ImageJ software (version 1.52a; NIH). Specifically for determining sumoylation levels, all SUMO signals (above the Ubc9-SUMO band, if it was present) were considered SUMO conjugates, and normalization was made to the corresponding signal from GAPDH immunoblots. Antibodies used for immunoblot analyses were: 1:500–1:1000 SUMO/Smt3 (y-84; Santa Cruz, sc-28649); 1:3000 GAPDH (Sigma, G9545); 1:500 Ubc9 (Santa Cruz, sc-6721); 1:3000 histone H3 (Abcam, ab1791); 1:1000 Myc epitope tag (Sigma, 05-724); 1:1000 Rpb3 (Abcam, ab202893); 1:5000 HA (12CA5; Sigma, 11583816001). The RPL3 antibody, which we used at a dilution of 1:200, was deposited to the DSHB by Warner, J. R. (DSHB Hybridoma Product ScRPL3 supernatant).
10
Protein extraction and Western blot analysis
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Cellular protein extraction occurred by resuspending cell pellets in 0.5 mL RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 1% SDS, 50 mM Tris-HCl pH 8) supplemented with PhosphataseArrest (G-Biosciences, Saint-Louis, MO, USA) and protease inhibitors (Complete Mini®, Roche), Basel, Switzerland). After 15 min incubation on ice with regular vortexing, samples were briefly sonicated (1 min, amplitude 30 kHz, pulse 1 s) and centrifuged at 13,200 rpm for 20 min at 4 °C. Solubilized proteins were transferred to new Eppendorf tubes and stored at −20 °C. Protein lysates were separated using Bis-Tris SDS-PAGE with a high-MW MOPS running buffer, and transferred onto nitrocellulose membranes (Hybond C, Amersham) using the Power Blotter System (Thermofisher, Waltham, MA, USA). Blocking the membranes for 1 h with blocking buffer (20 mM Tris-HCl, 140 mM NaCl, 5% BSA, pH 7.5) at RT was followed by overnight incubation with the primary antibody at 4 °C. Blots were then incubated for 1 h with the secondary, HRP dye-conjugated antibody (Dako, Glostrup, Denmark) after which chemiluminescent signals were detected with the Amersham Imager 680 (Cytiva, Marlborough, MA, USA) and quantified with the ImageJ software (v1.53j, National Institutes of Health, Bethesda, MD, USA) [94 (link)].
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