SDS-PAGE was carried out under denaturing and non-denaturing conditions. Under denaturing conditions, the purified protein was mixed with 2× sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue) containing 200 mM dithiothreitol (DTT) and boiled for 5 min at 95 °C. In contrast, non-denaturing SDS-PAGE was performed according to the protocol reported by Bender et al. (2005) (link). Briefly, the purified protein was mixed with 2× sample buffer containing 0.4% SDS (in the absence of a reducing agent) and loaded directly onto a gel (without boiling of the sample). The protein was separated by 5–20% polyacrylamide gradient gel (e-PAGEL, ATTO) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was incubated in 5% skim milk (Wako) in T-PBS buffer (phosphate-buffered saline, pH 7.2 (PBS) containing 0.05% Tween 20) for 60 min at 37 °C, and then incubated with a mAb against S1 protein in 5% skim milk in T-PBS buffer. Next, the membrane was incubated with a peroxidase-conjugated second antibody, goat anti-mouse IgG (H + L) (Jackson). The reacted protein was visualized using a TMB substrate kit (Invitrogen).
Skim milk
Manufactured by Fujifilm
Sourced in Japan, United States
Skim milk is a dairy product that is low in fat content. It is produced by removing the cream from whole milk, leaving behind a liquid with a low-fat concentration.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (8 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (5 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Imagequant las 4000 system, by GE Healthcare (3 mentions)
M per, by Thermo Fisher Scientific (3 mentions)
Can get signal solution 1, by Toyobo (3 mentions)
Can get signal solution 2, by Toyobo (3 mentions)
Immobilon p transfer membrane, by Merck Group (3 mentions)
Hybond, by Cytiva (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Fujifilm (2 mentions)
Skim milk, by Merck Group (2 mentions)
Szx16, by Olympus (2 mentions)
Wga conjugated horseradish peroxidase hrp, by Vector Laboratories (2 mentions)
2 mercaptoethanol, by Fujifilm (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Phosstop, by Roche (2 mentions)
57 protocols using skim milk
1
SDS-PAGE Analysis of Protein Samples
2
Astrocyte Differentiation Quantification
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Cells were washed with phosphate-buffered
saline (PBS) (Takara Bio, Shiga, Japan) twice and incubated with 4%
paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) at 4 °C
for 30 min. After PBS washing, the cells were incubated with PBS containing
5% skim milk (Fujifilm Wako Pure Chemical Corporation) and 0.2% Triton
X-100 (Thermo Fisher Scientific) at 4 °C for 30 min. Then, the
cells were treated with 5% skim milk solution containing primary antibody
anti-GFAP (an astrocyte marker, mouse monoclonal antibody, 1:500,
Merck Millipore) at 4 °C overnight. After washing with 5% skim
solution, the cells were incubated with 5% skim milk solution containing
secondary antibody Alexa Fluor 488-conjugated anti-mouse IgG (1:1000,
Thermo Fisher Scientific) at room temperature for 2 h. After washing
with 0.2% Triton X-100 solution, 0.2% Triton X-100 solution containing
Hoechst 33342 (1:1000; Dojindo, Kumamoto, Japan) was added to visualize
the nuclei, and the fluorescent cell images were obtained under the
microscope (IX71, Olympus Corporation, Tokyo, Japan). The obtained
images were analyzed by CellProfiler software,26 (link) and the ratio of the number of GFAP-positive cells to that
of the control was calculated as the rate of NSC differentiation into
astrocytes.
saline (PBS) (Takara Bio, Shiga, Japan) twice and incubated with 4%
paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) at 4 °C
for 30 min. After PBS washing, the cells were incubated with PBS containing
5% skim milk (Fujifilm Wako Pure Chemical Corporation) and 0.2% Triton
X-100 (Thermo Fisher Scientific) at 4 °C for 30 min. Then, the
cells were treated with 5% skim milk solution containing primary antibody
anti-GFAP (an astrocyte marker, mouse monoclonal antibody, 1:500,
Merck Millipore) at 4 °C overnight. After washing with 5% skim
solution, the cells were incubated with 5% skim milk solution containing
secondary antibody Alexa Fluor 488-conjugated anti-mouse IgG (1:1000,
Thermo Fisher Scientific) at room temperature for 2 h. After washing
with 0.2% Triton X-100 solution, 0.2% Triton X-100 solution containing
Hoechst 33342 (1:1000; Dojindo, Kumamoto, Japan) was added to visualize
the nuclei, and the fluorescent cell images were obtained under the
microscope (IX71, Olympus Corporation, Tokyo, Japan). The obtained
images were analyzed by CellProfiler software,26 (link) and the ratio of the number of GFAP-positive cells to that
of the control was calculated as the rate of NSC differentiation into
astrocytes.
3
Imaging cell wall structure in arbuscular mycorrhizal fungi
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The AMF cell walls were visualized with 3,3′-diaminobenzidine (DAB) staining [43 (link)]. Thalli (<2 mm) separated from soil granules were transferred to 2 mL centrifuge tubes and cleaned using 10% w/v potassium hydroxide at 80 °C in a preheated heat block for 8 min, then washed once with 0.5 mL methylene blue (0.1 mg/mL), five times with 1 mL water, and once with 1 mL phosphate-buffered saline (PBS; pH 7.5). Thalli were then immersed in 1 mL PBS containing 0.5% w/v skim milk (Wako, Osaka, Japan) and 0.4 μg mL−1 wheat germ agglutinin (WGA)-conjugated horseradish peroxidase (HRP; Vector, Burlingame, CA, United States). Thalli were incubated in this solution at room temperature for at least 8 h before being washed twice with 1 mL PBS, then were immersed in 1 mL PBS containing 0.2 mg mL−1 DAB tetrahydrochloride (Nakarai Tesque, Kyoto, Japan) and 0.1 μL mL−1 30% H2O2. Thalli were incubated in the DAB solution for at least 30 min at room temperature, then were soaked in 1 mL Tris-ethylenediaminetetraacetic acid (Tris-EDTA) buffer (TE buffer; 10 mM Tris-HCl, 1 mM EDTA; pH 8.0) to stop the HRP reaction. Images were obtained using a stereomicroscope (SZX16 or SZ61, Olympus, Japan) equipped with a charge-coupled device camera.
4
Aβ Protein Analysis in Mouse Brain
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On feeding day 58, the mouse brain cortex was homogenized with M-PER (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) containing 5% 2-mercaptoethanol (Wako, Osaka, Japan) at 75 °C for 5 min and loaded onto a 14% sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) (50 µg/lane). After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with 0.1% T-TBS containing 5% skim milk (Wako) at room temperature, the membrane was incubated with a mouse monoclonal anti-Aβ antibody (1:1000, clone 6E10, BioLegend, San Diego, CA, USA) or mouse monoclonal anti-GAPDH (1:10000, clone 5A12, Wako) in Can Get Signal solution 1 (Toyobo, Osaka, Japan) overnight at 4 °C. After washing with 0.1% T-TBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody against mouse IgG (1:2000; Cat. No. sc-2005, Santa Cruz) in Can Get Signal Solution 2 (Toyobo) for 2 h at room temperature. After washing, chemiluminescence on the membrane was detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) using an ImageQuant LAS 4000 system (GE Healthcare).
5
Western Blot Analysis of GFP Protein
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Worms were lysed in 20 mM Tris/HCl buffer (pH 7.4) containing 1.0 % Triton X-100, 150 mM NaCl, and cOmplete™ Protease Inhibitor Cocktail (F. Hoffmann-La Roche AG, Basel, Switzerland). After centrifugation at 12,000 rpm for 10 min, the supernatants were obtained. Protein concentration was measured using BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Supernatants containing 20 µg of proteins were separated by a 15 % SDS-PAGE gel under reducing conditions and blotted onto a PVDF membrane. The membrane was blocked with 5.0 % bovine serum albumin (Nacalai Tesque) for the detection of phosphorylated proteins and 1.0 % skim milk (Wako Pure Chemical Industries) for other proteins. The primary antibody was mouse anti-GFP IgG (1/1,000; Sigma-Aldrich Corporation, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG (1/4,000; MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan). Detection of the target proteins was carried out using West Pico Chemiluminescent Kit (Thermo Fisher Scientific) and LAS Image Analyzer (Fuji Film Corporation, Tokyo, Japan).
6
SARS-CoV-2 Spike Protein Binding Assay
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Recombinant human ACE2 proteins (R&D) were coated to Nunc maxisorp 96-well plates (Thermo Fisher Scientific, USA) at the concentration of 1 µg/mL, followed by incubation at 4°C overnight. The plates were washed 3 times with 1 × PBS containing 0.1% Tween 20 (PBST) and blocked with 5% skim milk (Wako, Tokyo, Japan) in PBST for 1h at room temperature. After being washed with PBST, the plates were added serial dilutions of the SARS2/SNFPP+CMP+TEV-H8STREPH6 proteins and incubated for 1h at room temperature, then washed 4 times with PBST. STREP tactin-HRP (IBA GmbH, Germany) was added to wells. After incubation for 1h at room temperature, the plates were washed 4 times with PBST and developed with 3,3’,5,5’-tetramethylbiphenyldiamine (TMB, Merck, USA). The reactions were stopped with 2M H2SO4, and the absorbance at 450 nm wavelength was measured by a Synergy HTX plate reader (BioTek Instruments, Highland Park, VT).
7
Polyclonal Antibody Production and Western Blot
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Immunization of a rabbit with the purified recombinant TbGMPR as an antigen, and preparation of the antiserum were performed by Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). Whole IgG was fractionated from the antiserum by ammonium sulfate precipitation followed by Ab-Rapid SPiN EX column chromatography (ProteNova Co. Ltd., Kagawa, Japan). Crude lysates of T. brucei were prepared in RIPA buffer and subsequent centrifugation. The lysates were separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad). The membrane was then incubated overnight at 4°C with 5 μg/ml of the anti-TbGMPR polyclonal IgG in Tris-buffered saline with 0.05% Tween-20 (TBST) containing 5% skim milk (Wako). After having been washed with TBST, the blots were incubated with anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technologies, Danvers, MA). The immunoreactivities were visualized by using ImmunoStar Zeta (Wako) and LAS4000 imaging device (GE Healthcare Japan).
8
Western Blot Analysis of TGF-β Signaling Pathway
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Whole‐cell lysates from mouse hearts were washed in PBS and lysed using CelLytic MT Cell Lysis Reagent (Sigma‐Aldrich, St. Louis, MO) containing Complete Protease Inhibitor Cocktail and PhosSTOP (Roche Applied Science, Indianapolis, IN). The membranes were blocked with skim milk (WAKO, Osaka, Japan) or Block Ace Powder (DS Pharma, Osaka, Japan). The following primary antibodies were used: Smad2 (1:1000 dilution), Phospho‐Smad2 (Ser465/467) (1:1000 dilution), Smad3 (1:1000 dilution), Phospho‐Smad3 (Ser423/425) (1:1000 dilution), IRF5 (1:1000 dilution; all Cell Signaling Technology, Danvers, MA), and GAPDH (1:1000 dilution; Santa Cruz Biotechnology, Inc, Santa Cruz, CA). Band density was analyzed using Image J software. The densitometric values (4‐8 biological replicates with 3 or more technical replicates each) were normalized using a GAPDH signal.
9
Western Blot Analysis of Cellular Proteins
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The HeLa and NCI-H1975 cells were collected and lysed in a RIPA buffer (Thermo Fisher Scientific, Carlsbad, CA) with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and subjected to two freeze–thaw cycles. The lysed cells were centrifuged at 15,000×g for 20 min at 4°C, and the supernatants were collected as protein samples. Laemmli’s sample buffer (Bio-Rad, Hercules, CA) was added to the supernatants, and the mixture was boiled for 5 min. Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad) at 60 V in a transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) for 60 min at 4°C. The membrane was probed overnight with specific antibodies diluted with TBST (10 mM Tris-HCl [pH 7.4], 0.1 M NaCl and 0.1% Tween-20) containing 5% skim milk (Wako Pure Chemical Industries, Osaka, Japan) at 4°C. After probing with HRP-conjugated secondary antibodies, the bound antibodies were detected with an Immobilon® western HRP substrate (MilliporeSigma, Burlington, MA). Densitometry was performed using Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan).
10
Western Blot Protein Detection Protocol
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After the electrophoresis, proteins separated on the gel were transferred onto a nitrocellulose membrane (pore size 0.2 µm, Bio-Rad, Hercules, CA, USA) for 120 min using the Trans-blot SD semi-dry electrophoretic transfer cell (Bio-Rad) at 70 V (voltage constant). The membrane was blocked for 1 h at room temperature with 5% skim milk (Wako Pure Chemical Industries) in Phosphate buffered saline containing 0.1% Tween-20 (PBS-T). The membrane was then incubated for 8 h at 4 °C with a primary antibody, mouse anti-His tag antibody (Cell Signaling Technologies, Danvers, MA, USA), at 1:1000 dilution in PBS-T containing 5% skim milk. After washing three times with PBS-T for 15 min, the membrane was incubated for 1 h at room temperature with a secondary antibody, HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technologies), diluted by 1:1000 ratio in PBS-T containing 5% skim milk. The membrane was washed three times with PBS-T for 15 min and then treated with Western Blot detection solution A and B of Pico EPD Kit (ELPIS Biotech, Daejeon, South Korea) by 1:1 ratio for visualizing protein bands. Photographs of blots were taken by the Chemidoc XRS+ illumination system (Bio-Rad).
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