Cells lysates were prepared in RIPA buffer (Cell signaling, Danvers, USA). Equal amounts of protein lysates were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked at room temperature for 1 h with 5% blotto (Santa Cruz, Dallas, USA) followed by exposure to Duox-1 antibody (1:500 dilution in 5% blotto) overnight at 4 °C. After washing with TBS plus 1% Tween-20, membranes were incubated with 1:5000 diluted goat anti rabbit IgG-HRP antibody for 1 h at room temperature. Membranes were incubated with Pierce Western Blotting Substrate (Pierce, Rockford, USA) and imaged. Antibody to GAPDH (Abcam, Cambridge, USA) was used as a loading control.
Blotto
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Blotto is a laboratory reagent used for blocking non-specific binding in immunoassays. It is a solution of non-fat dry milk or other proteins that can be used to reduce background signals and improve the specificity of antibody-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx imaging system, by LI COR (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Tween 20, by Merck Group (2 mentions)
Mouse anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Antibody to gapdh, by Abcam (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Fluorescently conjugated secondary antibodies, by LI COR (1 mentions)
Criterion tgx precast gel, by Bio-Rad (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Pefabloc sc, by Merck Group (1 mentions)
Image studio lite ver 5, by LI COR (1 mentions)
Immobilon, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Sc 2325, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Cdp star, by Thermo Fisher Scientific (1 mentions)
Stat 1 tyr701, by Cell Signaling Technology (1 mentions)
15 protocols using blotto
1
Western Blot Analysis of Duox-1 Protein
2
Western Blot Analysis of Protein Markers
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Protein extracts were mixed with 4× sample buffer (8% SDS, 20% v/v glycerol, 0.002% bromphenolblue, 62.5 mM Tris-Cl, pH 6.8) supplemented with 100 mM dithiothreitol (DTT) and incubated for 10 min at 95°C. Proteins were separated on 4–15% Criterion TGX precast gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Membranes were blocked using 5% non-fat dry milk (Blotto, Santa Cruz Biotechnologies) in TBS, 0.1% v/v Tween® 20 (Merck) for 30 min to 1 h at room temperature. Primary antibody incubations were performed overnight at 4°C in blocking buffer. The following primary antibodies were used: rabbit anti-PTPA (Cell Signaling Technologies, 3330, 1:1000 dilution), mouse anti-PTPA (BioLegend, 824401, 1:1000 dilution), mouse anti-PP2A C (Sigma-Aldrich, 05-421, 1:1000 dilution), rabbit anti-LC3B (Novus, NB100-2220, 1:1000 dilution), rabbit anti-GADD34 (Proteintech, 10449-1-AP, 1:1000 dilution), mouse anti-vinculin (Santa Cruz Biotechnology, V284, 1:2000 dilution). After washing in TBS, 0.1% v/v Tween® 20, blots were incubated for 1 h at room temperature with fluorescently conjugated Alexa Fluor Plus 800 donkey anti-mouse and 700 donkey anti-rabbit (both from ThermoFisher) secondary antibodies. After washing in TBS, 0.1% v/v Tween® 20, blots were imaged and analysed using the Odyssey CLx imaging system (LI-COR Biosciences).
3
Western Blot Protein Analysis Protocol
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Protein lysates were obtained after washing the cells with PBS and subsequently adding protein lysis buffer [50 mM Tris–Cl (pH 7.4), 100 mM NaCl, 1.0% IGEPAL® CA-630 (all from Sigma-Aldrich)] containing protease inhibitors Complete® and Pefabloc® SC (both from Merck). Lysates were snap frozen, thawed on ice and cleared by centrifugation at 13,000 RPM for 10 min at 4 °C. Lysates were mixed with 4 × sample buffer [8% SDS, 20% v/v glycerol, 0.002% bromophenolblue, 62.5 mM Tris–Cl (pH 6.8)] supplemented with 100 mM dithiothreitol (DTT) and incubated for 10 min at 95 °C. Proteins were separated on 4–15% Criterion TGX precast gels (Bio-Rad), and transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Blots were blocked using 5% non-fat dry milk (Blotto, Santa Cruz Biotechnologies) in PBS for 1 h at RT. Primary antibody incubations were performed overnight at 4 °C in blocking buffer. After washing in PBS, 0.1% v/v TWEEN® 20, blots were incubated for 1 h at RT with fluorescently conjugated secondary antibodies (LI-COR Biosciences or Jackson ImmuneResearch). After washing in PBS, 0.1% v/v TWEEN® 20, blots were imaged using the Odyssey CLx Imaging system and analysed with Image Studio™ Lite Ver 5.2 (both from LI-COR Biosciences).
4
Western Blot Protein Analysis Protocol
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Monolayers and spheroids were lysed in boiling lysis buffer (2.5% SDS, Tris-HCl 250 mM pH 7.4) and protein concentration was determined using a colorimetric assay (DC protein assay from BioRad, Hercules, CA, USA). 30 μg of protein was supplemented with Laemmli sample buffer and separated on an SDS-PAGE gel. Proteins were transferred to PVDF membranes (Immobilon, Millipore, Billerica, MA, USA) and blocked with 5% Blotto (Santa Cruz Biotechnology, Dallas, TX, USA) in PBS. Primary antibodies were diluted in 5% non-fat dry milk or 5% BSA. The following primary antibodies were used: anti-ASS1 (clone 25, 1:500, BD Biosciences, San Jose, CA, USA; clone 2B10, 1:500, Sigma-Aldrich, St. Louis, MO, USA; clone 2C10, 1:500, Millipore, Billerica, MA, USA), anti-α-tubulin (#A5441, 1:10,000, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were from BioRad (Hercules, CA, USA). Chemiluminescence was detected with the enhanced SuperSignal West Pico Substrate (Pierce, Rockford, IL, USA) with a Biospectrum Imaging System (UVP, Upland, CA, USA).
5
SARS-CoV-2 Antibody Binding ELISA
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The antibodies purified from a 1 mL transient transfection were tested for their binding against SARS CoV-2 S1 (Sino#40591-V08H) and RBD (Acro#SPD-C52H3) proteins using an ELISA. Briefly, 2 μg/mL of either SARS CoV-2 RBD or S1 protein were coated onto 96-well ELISA plates (Nunc Maxisorp) and left overnight at 4 °C. The wells were blocked with 5% Blotto (Santa Cruz) in 1× PBST for 1 h at room temperature. After rinsing the plates three times, 1:100 and 1:10,000 dilutions of the purified recombinant antibody solution were added to the plates, which were incubated on a rocker platform for 2 h at room temperature. After rinsing the plates three times with 1× PBST, a rabbit anti-human IgG conjugated to horseradish peroxidase (Jackson Immuno Research) was added to each well. The plates were incubated for 1 h at room temperature followed by washing with 1× PBST and the addition of the TMB substrate. The reaction was stopped by adding 1 N of sulfuric acid and the absorbance was read at 405 nm.
6
TSPO Protein Expression Analysis
7
Microglial Protein Expression Analysis
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Following treatment, microglial culture media were removed and cells were lysed with the 2x Laemmli sample buffer containing 5% 2-mercaptoethanol. The samples were then heated at 100°C for 5 min and vortexed before being loaded onto 4–15% precast acrylamide gels for electrophoresis. Gels were transblotted onto nitrocellulose membranes (0.45 µm), rinsed in TTBS (Tris-HCl with NaCl and Tween 20), and blocked with 5% Blotto (Santa Cruz, Dallas, TX) for 1 h at RT. Membranes were then probed with rabbit anti-β-actin (#4970) and rabbit anti-phospho-Stat3(Tyr705) (#9131) or -Stat1(Tyr701) (#9171) Abs (Cell Signaling Technology, Danvers, MA) overnight at 4°C. After washing 3x with TTBS, alkaline phosphatase (AP) conjugated secondary antibody (1 : 5,000 in 1% Blotto, Promega, Madison, WI) was added to the membranes at RT for 1 h. Following 3x washing with TTBS, membranes were rinsed in the assay buffer (1x) twice (2 min each) and then incubated in the substrate solution (CDP-Star, Applied Biosystems, Foster City, CA) for 10 min followed by imaging using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).
8
Western Blot Protein Analysis Protocol
9
Western Blot Analysis of Cell Signaling Proteins
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Following treatments, cells were washed twice with PBS and lysed in 1× SDS sample buffer. Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were washed twice with tween Tris-buffered saline before blocking nonspecific binding with 5% nonfat dry milk (Blotto, Santa Cruz Biotechnology, Dallas, TX, USA) or 3% bovine serum albumin. Anti-Poly ADP-ribose Polymerase (PARP) (11835238001-Roche) antibody, that detects total and cleaved PARP was used at 1:1000 dilutions, and membranes were incubated for 2 h at room temperature. Anti-Cyclin D1 (C7464, Sigma Aldrich) and anti-CDK4 (SAB140559, Sigma Aldrich) were detected similar to PARP protein. Membranes were washed three times, and detection was performed using horseradish peroxidase-conjugated secondary antibody as described previously [40 (link)].
10
Western Blot Analysis of Drosophila Proteins
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Heads from 1 day old flies were homogenized in 2× SDS-PAGE sample buffer followed by boiling at 95°C for 5 min. Samples were separated using SDS-PAGE and electro blotted onto nitrocellulose membrane (Hybond-C Extra; GE Healthcare) using semidry transfer assembly (Bio-Rad). Following blocking with 5% Blotto (Santa Cruz Biotechnology, CA), blots were incubated overnight at 4°C in appropriate dilutions of primary antibodies [anti-α-tubulin (1:5000 dilution; E7 DSHB), anti-Gαq (1:1000 dilution), anti-TRP (1:5000 dilution), anti-Rh1 (1:200,4C5 DSHB), anti-INAD (1:2000) and anti-NORPA (1:1000)]. Protein immunoreacted with the primary antibody was visualized after incubation in 1:10,000 dilution of appropriate secondary antibody coupled to horseradish peroxidase (Jackson Immuno Research Laboratories) for 2 h at room temperature. Blots were developed with ECL (GE Healthcare) and imaged using a LAS 4000 instrument (GE Healthcare).
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