Again, ≈50 mg of muscle tissue were mechanically homogenized (6000 rpm for 2 × 30 s) in 1 × CST Cell Lysis Buffer (Cell Signaling, Frankfurt am Main, Germany) using Precellys ceramic beads. Samples were incubated on ice for 20 min and diluted 1:2 with 2× Laemmli (31.25 mM tris(hydroxymethyl)aminomethane, 1% sodium dodecyl sulfate, 5% glycerol). After denaturation (10 min, 94 °C) and centrifugation (2 min, 21,000× g, 4 °C), the protein concentration was also determined using the bicinchoninic acid method described earlier. The protein concentrations were then adjusted to 1 mg/mL with 1 × Laemmli, and beta-mercaptoethanol was added (to 0.4% finally). The samples (20 µg total protein) were separated by SDS-PAGE (Bio-Rad TGX Stain-Free FastCast Acrylamide kit; Bio-Rad Laboratories GmbH, Munich, Germany). After electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by semi-dry blotting (60 min, current 80 mA/gel).
Tgx stain free fastcast acrylamide kit
Manufactured by Bio-Rad
Sourced in United States, Germany
The TGX Stain-Free FastCast Acrylamide Kit is a ready-to-use solution for the preparation of polyacrylamide gels. It is designed for the separation and analysis of proteins using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Image lab software, by Bio-Rad (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Bradford protein assay kit, by Bio Basic (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Cst cell lysis buffer, by Cell Signaling Technology (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Cst 9271, by Cell Signaling Technology (1 mentions)
Image lab ver 6, by Bio-Rad (1 mentions)
Il 17a, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
30 protocols using tgx stain free fastcast acrylamide kit
1
Muscle Protein Extraction and Quantification
2
Western Blot Detection of Phosphorylated AKT
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For electrophoresis, Bio-Rad TGX Stain-Free FastCast Acrylamide-Kit (Bio-Rad Laboratories GmbH, Munich, Germany) gels were used. All gels were blotted on polyvinylidene fluoride membranes (PVDF, pore size 0.45 µm, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), blocked for 1 h with 3% powdered milk in PBS and incubated overnight with a primary antibody for AKT phosphorylated at serine 473 (CST #9271, dilution 1:1000, Cell Signaling Technologies). After washing, the membranes were incubated for 2 h with a secondary antibody (antirabbit IgG HRP, CST #7074, dilution 1:2000, Cell Signaling Technologies). Bands were visualized using Lumigen ECL Ultra (Lumigen Inc., Southfield, MI, USA) in a Bio-Rad Chemi-Doc MP system (Bio-Rad Laboratories GmbH). The images were analyzed using Image Lab Ver. 6.0.1 software (Bio-Rad Laboratories GmbH, Hercules, CA, USA) and normalized for total protein concentration. The molecular weight of phosphorylated human AKT is predicted to be 56–57 kDa.
3
Western Blot Analysis of Conjunctival Proteins
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A Western blot analysis was performed using standard techniques. In brief, conjunctivas were homogenized in RIPA lysis buffer with PMSF (RIPA: PMSF = 100: 1) (Beyotime, Shanghai, China), to extract total protein. After being quantified by a BCA protein assay kit (Beyotime), protein samples were separated using a 12% TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, Hercules, CA) and blotted to PVDF membranes (Merck-Millipore, Darmstadt, Germany). Membranes were blocked with 5% non-fatty milk and incubated with primary antibodies against GAPDH (1:5000, Cell Signaling Technology, Danvers, MA), IL-17A (1:1000, Abcam, Cambridge, UK). Membranes were then washed, incubated with appropriate peroxidase-conjugated secondary antibodies, and developed into an ECL kit (Najm Biotech ECL, Tehran, Iran).
4
Western Blot Analysis of YY1 and GAPDH Proteins
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Proteins from tissue and cell lines were extracted using a total protein extraction kit (BestBio). The protein concentration was determined using Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific) and after incubation for 1 hour, the protein concentration of samples and standards was determined at a wavelength of 562 nm using a microplate reader. All proteins were standardized to a concentration of 3 μg/μL and denatured at 99°C for 5 minutes. Total proteins were separated using TGX Stain‐Free™ FastCast™ Acrylamide Kit (BioRad) and transferred to the polyvinylidene difluoride membranes (PVDF, 16 20 177, BioRad). Subsequently, the membranes were blocked with 5% Bovine Serum Albumin (BSA) for 3 hours and incubated with primary antibody YY1 (1:12 000, Abcam) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, 1:5000, ZenBio) overnight at 4°C. The membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody goat anti‐rabbit (1:10 000, HuaBio) and goat anti‐mouse (1:2000, HuaBio) for 1 hour at room temperature. Lastly, Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used to detect the chemiluminescence intensity via the ChemiDoc™ MP Imaging System (BioRad).
5
Protein Extraction and Western Blot Analysis
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Protein extraction was performed with 100 μL RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China) by using the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, 1610182, Hercules, CA, USA). The extracted proteins were separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a semidry transfer membrane. The membrane was sealed with a sealing solution (Bio-Rad, P0023B, Shanghai, China) at 25 °C for 2 h and then incubated with the following antibodies at 4 °C for 12 h: purified anti-Vg antibody (1:1000), anti-30Kc19 antibody (1:1000), anti-V5 tag antibody (1:1000, AB_2533339, Thermo Fisher, Waltham, MA, USA), and anti-α-tubulin antibody (1:5000, T0033, Affinity). After three times washing with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST; pH 7.5), the membrane was incubated with HRP-conjugated anti-mouse IgG (1:5000, Bioworld Technology, St Louis Park, MN, USA) at 37 °C for 2 h. After washing the membrane again with TBST three times, an appropriate amount of ECL (Bio-Rad) coloring solution (1:1) was added, and the membrane was kept incubated in dark. Image Lab software was used to process and analyze the generated images.
6
Adenosine Kinase Expression Quantification
7
Immunoblotting analysis of apoptotic proteins
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The immunoblotting experiment was performed as previously described [19 (link),20 (link)]. In brief, total protein was isolated from tissue samples using RIPA lysis buffer with protease inhibitor cocktail tablets (Roche, Switzerland), and quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, U.S.A.). The total protein samples were loaded and separated on TGX Stain-Free™ FastCast™ Acrylamide Kit (Bio-Rad, U.S.A.) and transferred to PVDF membranes (Merck Millipore, Germany). The membranes were blocked with 5% skim milk for 2 h and incubated with primary antibodies against cathepsin D (Abcam, U.S.A., 1:2000), Bax (ProteinTech, China, 1:200), cathepsin B and Bcl-2 (Cell Signaling Technology, U.S.A., 1:1000) overnight at 4°C, which was followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. Signals were visualized by enhanced chemiluminescence (ECL) reagents (Abvansta, U.S.A.) and captured by a Chemi DocMP Imaging System (Bio-Rad, U.S.A.). Total protein was used for normalization. Immunoreactive bands were quantified using ImageJ.
8
Optimized Western Blot Protocol
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Samples for western blot were homogenized by a tissue lyser in
radioimmunoprecipitation assay (RIPA) buffer containing complete protease
inhibitor cocktail (Merck, Darmstadt, Germany) then centrifuged at
10,000 × g for 10 min to remove cellular debris.
Equivalent amounts of protein (20 µg) and the protein marker (NZYColour Protein
Marker II, MB090, NZYtech, Lisbon, Portugal) were separated on 12% acrylamide
gels (TGX Stain-Free™ FastCast™ Acrylamide kit; Bio-Rad, Hercules, CA, USA)
gels. This technology contains trihalo compounds that react with tryptophan
residues in a UV-induced reaction to produce fluorescence that can be detected
by the ChemiDoc imaging system. Total protein was detected using the ChemiDoc
imager in the membrane after transfer onto nitrocellulose membranes. Membranes
were blocked for 1 h with bovine serum albumin (BSA) 5% and then incubated with
primary antibody [H3K27me from Cell Signaling (9733) at 1:1000 dilution in 5%
BSA; Cell Signalling Technology, Beverly, MA, USA]. The membranes were incubated
for 1 h at room temperature with secondary antibody horseradish
peroxidase-conjugated anti-rabbit (1:1000). Detection was conducted using
SuperSignal West Femto chemiluminescent substrate kits (Pierce Biotechnology
Inc., Rockford, IL, USA) using the ChemiDoc imager.
radioimmunoprecipitation assay (RIPA) buffer containing complete protease
inhibitor cocktail (Merck, Darmstadt, Germany) then centrifuged at
10,000 × g for 10 min to remove cellular debris.
Equivalent amounts of protein (20 µg) and the protein marker (NZYColour Protein
Marker II, MB090, NZYtech, Lisbon, Portugal) were separated on 12% acrylamide
gels (TGX Stain-Free™ FastCast™ Acrylamide kit; Bio-Rad, Hercules, CA, USA)
gels. This technology contains trihalo compounds that react with tryptophan
residues in a UV-induced reaction to produce fluorescence that can be detected
by the ChemiDoc imaging system. Total protein was detected using the ChemiDoc
imager in the membrane after transfer onto nitrocellulose membranes. Membranes
were blocked for 1 h with bovine serum albumin (BSA) 5% and then incubated with
primary antibody [H3K27me from Cell Signaling (9733) at 1:1000 dilution in 5%
BSA; Cell Signalling Technology, Beverly, MA, USA]. The membranes were incubated
for 1 h at room temperature with secondary antibody horseradish
peroxidase-conjugated anti-rabbit (1:1000). Detection was conducted using
SuperSignal West Femto chemiluminescent substrate kits (Pierce Biotechnology
Inc., Rockford, IL, USA) using the ChemiDoc imager.
9
Protein Detection and Quantification using BCA
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The total amount of protein was detected by the bicinchoninic acid method (Beyotime, Shanghai, China). Proteins (20 μg) were separated by SDS-PAGE on 10% polyacrylamide gels and transferred electrophoretically to polyvinylidene fluoride membranes with the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, California, USA). The membranes were blocked with 5% nonfat dry milk in TBS + 0.1% Tween-20 for 1 h and incubated overnight at 4 °C with the primary antibodies in bond primary antibody diluent (see Supplementary Table 2, Supplemental digital content 1, http://links.lww.com/WNR/A654 , for antibody information), followed by secondary antibodies [horseradish peroxidase (HRP)–labeled goat antimouse immunoglobulin (Ig)G, 1:5000, and HRP-labeled goat antirabbit IgG, 1:10 000, Abcam, Cambridge, UK] at room temperature for 1 h. The proteins were detected by the Bio-Rad ChemiDoc Touch Image System (Bio-Rad).
10
Protein Separation via SDS-PAGE
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Samples were separated on a polyacrylamide gel; the procedure was shortened to SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis), which is a standard technique for separating proteins according to their molecular weight. Polyacrylamide gels were performed using the TGX Stain-Free™ FastCast™ Acrylamide Kit (Bio-Rad Laboratories, Inc.).
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