Cells were washed twice with PBS and suspended in lysis buffer (Beyotime Institute of Biotechnology) for 30 min on ice. Lysates were centrifuged at 13,000 × g for 10 min at 4°C and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, USA) prior to 10% SDS-PAGE. Membranes were then blocked with 5% bovine serum albumin (Biosharp, Hefei, China) in Tris-buffered saline (Beyotime Institute of Biotechnology) containing Tween-20 (TBST; Sangon Biotech Co., Ltd., Shanghai, China) for 1 h at room temperature. After three washes with TBST, membranes were incubated with primary antibodies at 4°C overnight. Membranes were then washed three times with TBST prior to incubation with secondary antibody (cat. no. E030120; 1:1,000; EarthOX Life Sciences, Millbrae, CA, USA) for 2 h at room temperature. The protein bands were exposed in a dark room and analyzed using AlphaView SA 3.4.0. software (ProteinSimple, San Jose, CA, USA). Protein expression was normalized to GAPDH.
Tris buffered saline (tbs)
Manufactured by Beyotime
Sourced in United States
Tris-buffered saline is a commonly used buffer solution that maintains a stable pH environment. It is composed of tris(hydroxymethyl)aminomethane (Tris) and sodium chloride (NaCl) dissolved in water. The primary function of this buffer is to maintain a consistent pH, typically around 7.4, which is essential for various biological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Beyotime (2 mentions)
Bovine serum albumin (bsa), by Biosharp (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti rabbit igg, by Beyotime (1 mentions)
Non fat dry milk, by Beyotime (1 mentions)
Imagequant tl 7, by GE Healthcare (1 mentions)
Bca quantitation kit, by Thermo Fisher Scientific (1 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l, by Beyotime (1 mentions)
Donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Roche (1 mentions)
Tween 20 tbst, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Anti icam 1, by Santa Cruz Biotechnology (1 mentions)
8 protocols using tris buffered saline (tbs)
1
Western Blot Analysis of Protein Expression
2
MCL Induces Apoptosis and EMT Modulation
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U251MG cells (1×105) were treated with MCL (0, 5, 10 and 15 µM) for 48 h at 37°C, and then total protein was extracted by RIPA lysis buffer and quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein (30–50 µg) was separated via 10–12% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane and blocked with 5% non-fat dry milk (Beyotime Institute of Biotechnology) for 1h at room temperature. The membranes were then incubated at 4°C overnight with the following primary antibodies: Anti-cleaved caspase-3 (1:1,000), anti-cleaved caspase-9 (1:1,000), anti-COX-2 (1:1,000), anti-p-IκBα (1:500), anti-IκBα (1:1,000), anti-β-actin (1:2,000), anti-Bcl-2 (1:2,000), anti-Bax (1:2,000), anti-Vimentin (1:1,500), anti-MMP-9 (1:800) and anti-N-cadherin (1:500). Membranes were washed three times with 1X Tris-buffered saline with 0.1% Tween (Beyotime Institute of Biotechnology) and incubated with the anti-rabbit IgG (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse IgG (1:5,000; cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology) and semi-quantification was performed using ImageQuant TL 7.0 software (GE Healthcare).
3
Quantifying Intestinal Mucin and Tight Junctions
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The protein expression of mucin 2 (MUC-2) and tight junction proteins, including claudin-1, in the jejunum tissue was determined by Western blotting. Total protein extraction was performed using Tissue Protein Extraction Reagent (Thermo Pierce, 78510), and protein quantification was then performed using the BCA Quantitation Kit. After SDS-PAGE and membrane transfer, Tris-buffered saline (containing 5 % non-fat dry milk or bovine serum albumin) (Beyotime Biotechnology) was added to the membrane for blocking at room temperature for 1 h. The antibody (1:100) (Beyotime Biotechnology) was then added and incubated overnight at 4°C, followed by washing the membrane. Secondary antibody (goat anti-Mouse IgG (H + L)) (Beyotime Biotechnology) was added and incubated at room temperature for 1 h and then washed. SuperSignal® West Dura Extended Duration Substrate was used for Western blot detection. The optical densities of the bands were analysed using Image J software. β-Actin was used as an internal control and was found to exhibit no differences between groups. The relative abundance of each target protein was expressed as the ratio of target protein:β-actin.
4
Immunofluorescence Assay of γδT Cells
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Immunofluorescence assays of the purified peripheral γδT cells were carried out by Biossci Company. Blocking was performed in Tris‐buffered saline (Beyotime) with 10% normal serum (Biological Industries) and 0.1% Triton X‐100 (Beyotime). The 1:50 diluted rabbit anti‐human CD206 antibody (Abcam) and the 1:400 diluted donkey anti‐rabbit secondary antibody (Life Technologies) were used for the immunofluorescence assay. DAPI dye (Gibco) was used at room temperature for 20 min at a 1:500 dilution to identify the nucleus. The images were aquired on an Olympus BX53 microscope.
5
Western Blot Analysis of c-kit and SCF Protein Expression
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The protein expression of c-kit and SCF was determined by western blot analysis, with modifications made to previously described methods (He et al., 2017; (link)Li et al., 2021) (link). Specifically, frozen colonic tissue (100 mg) was homogenized in protein lysate (1 mL) containing a protease inhibitor cocktail (1:200). The homogenate was centrifuged at 13,000 × g for 5 min, and the supernatant was collected to determine the protein concentration using the bicinchoninic acid protein assay kit (Roche). Equal amounts of protein samples (40 µg) were separated by 10% SDS-PAGE (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST; Beyotime) at 37°C for 2 h. We used GAPDH as an internal reference. The membranes were incubated overnight at 4°C with the primary antibody (1:1,000). After washing in TBST, the secondary antibody (1:5,000) was added and incubated at 25°C for 30 min. The blots were subsequently washed with TBST and exposed to enhanced chemiluminescence-plus reagents for chemiluminescence detection. The grayscale of the target bands was analyzed using Image-pro Plus 6.0 software (Media Cybernetics Inc.).
6
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed using cell lysates, as described in our previous study (Wu et al. 2014) (link). Briefly, proteins were collected from cell lysates, by centrifuging at 12,000 g at 4°C for 15 min, with concentration measured using Bradford assay. After diluting in loading buffer and heating at 95°C for 5 min, the samples were subjected to electrophoresis on SDS-PAGE gel at 120 V. After electrophoresis, the proteins were transferred to nitrocellulose membranes, blocked in blocking buffer (5% milk and 0.5% BSA) for 1 h and washed 3 times with Trisbuffered saline containing 0.05% Tween 20 (Beyotime Biotechnology, Shanghai, PRC). The membranes were incubated with primary antibodies overnight, washed as aforementioned and reacted with secondary horseradish peroxidase-conjugated antibodies at room temperature for 1 h. The primary antibodies used were anti-Ac-P53 (Abcam; 1:500), anti-GAPDH (Santa Cruz Biotechnology, 1:3000), anti-ICAM-1 (Santa Cruz Biotechnology; 1:500), anti-P53 (Cell Signaling Technology; 1:1000) and anti-SIRT1 (Abcam; 1:1000).
7
Protein Extraction and Western Blot Analysis
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Cells were lysed in 1 ml cell lysate (Total Protein Extraction kit; ProMab) for 30 min at 4°C. The cell lysates were centrifuged at 12556 × g for 30 min at 4°C to remove insoluble material. The resulting supernatant was frozen at −80°C for later analyses by SDS-PAGE and immunoblotting. A total amount of 25 μl cell lysate were resolved on 12% SDS-PAGE gels (Sigma), followed by electrophoretic transfer onto polyvinylidene fluoride membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were blocked with 5% non-fat milk blocking buffer and then incubated overnight at 4°C with primary antibody (1:500 dilution; Abcam). Following extensive washing with Tris-buffered saline (TBS) containing 0.5% Tween 20 (Beyotime Institute of Biotechnology), membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:4,000 dilution; Thermo Pierce, Rockford, IL, USA) at room temperature for 1 h, followed by additional washes with TBS containing 0.5% Tween 20. The blots were developed using a chemiluminescence kit. Densitometric analyses of autoradiographic bands were normalized to GAPDH expression, taking into account the size and area of the bands (Scion Image software (Scion Corp., Frederick, MD, USA).
8
Western Blot Analysis of CRMP-5 and β-Actin
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Western blot analysis was performed as previously described (24 (link)). Briefly, lysates were separated using 8, 10 and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, Jiangsu, China) and were then electrophoretically transferred to a polyvinylidene difluoride membrane (Beyotime Institute of Biotechnology). Membranes were blocked in Tris-buffered saline (TBS; Beyotime Institute of Biotechnology) with 5% milk and 0.05% Tween-20 and were then probed with the following primary antibodies at 4°C overnight: Rabbit anti-CRMP-5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, cat. no. sc-292382; 1:1,000) and mouse anti-β-actin (cat. no. A5441; Sigma-Aldrich; 1:1,000). Subsequent to washing with TBS + 0.05% Tween; the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (cat no. 115-001-008) or anti-rabbit secondary antibodies (cat no. 711-001-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:5,000) and were then visualized using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology).
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