Protein was obtained from bovine preadipocytes using a protein extraction kit, supplemented with PMSF (Beyotime, Shanghai, China), and protein concentration was quantified with the Quantitative Protein Kit (the BCA method) (APPLYGEN, Beijing, China). For Western blot, 20 μg total cellular protein was separated on a 10% polyacrylamide gel, and then transferred onto PVDF. After blocking in Quick Block™ Western blocking solution (Beyotime, Shanghai, China) for 30 min at RT, the PVDF was sequentially incubated overnight at 4 °C with primary antibodies, respectively. Table 1 lists the dilution factor and company name of different antibodies. After washing in TBST-1× buffer 3 times (10 min each time) (Sangon Biotech Co., Ltd., Shanghai, China), the Goat anti-rabbit HRP antibody (absin, 1:10,000) diluted in Secondary Antibody Dilution Buffer (Beyotime, Shanghai, China) was incubated for 2 h, and washed in TBST-1× buffer.
Quickblock western blocking solution
Manufactured by Beyotime
Sourced in China
QuickBlock™ Western blocking solution is a ready-to-use buffer designed for blocking non-specific binding in Western blot analysis. It is a pre-formulated solution that helps to reduce background signal and improve the signal-to-noise ratio during the blocking step of the Western blotting procedure.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Ecl working solution, by Bio-Rad (2 mentions)
0.45 um pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Secondary antibody dilution buffer, by Beyotime (1 mentions)
Quantitative protein kit, by Applygen (1 mentions)
M per r mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Loading buffer, by Ipsen (1 mentions)
Trib3, by Proteintech (1 mentions)
Hrp conjugated anti mouse igg, by Proteintech (1 mentions)
Anti rabbit igg, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Western primary antibody diluent, by Beyotime (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Proteinase and phosphatase inhibitors, by Beyotime (1 mentions)
9 protocols using quickblock western blocking solution
1
Bovine Preadipocyte Protein Extraction and Western Blot
2
Western Blot Protein Extraction and Detection
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The cells were gathered and broken down in M-PER (R) Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) while being kept on ice for a duration of 30 min. The cell lysates underwent resolution through SDS-PAGE and were subsequently transferred to PVDF membranes (Cytiva, USA). The PVDF filters were treated with QuickBlock™ Western blocking solution (Beyotime, China) for 1 h while gently shaking. Subsequently, the filters were incubated with the specified antibodies at 4 °C overnight. Goat secondary antibodies conjugated with HRP (1:1000, Beyotime, China) were employed. After being washed with TBST, the membranes underwent a thorough cleansing, followed by incubation of the secondary antibody at room temperature for 2 h. To examine the protein bands, we employed a chemiluminescent western blot detection method.
3
Western Blot Analysis of TRIB3 Protein
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After 48 h of cell transfection in a 6-well plate, the cells were lysed on ice for 30 min using RIPA buffer (Beyotime, Shanghai, China), which included protease inhibitors and phosphatase inhibitors (BOSTER, Wuhan, China). The lysate was then centrifuged to obtain the supernatant. Subsequently, the protein concentration in the samples was determined using a BCA protein assay kit (CWBIO, Jiangsu, China). The protein samples were mixed with loading buffer (Epizyme, Shanghai, China), heated at 95 °C for 10 min to denature the proteins, and then loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. The proteins were subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After a 20 min incubation in a QuickBlock™ Western blocking solution (Beyotime, Shanghai, China), primary antibodies and HRP-conjugated secondary antibodies were added sequentially to incubate the membrane. Finally, the target proteins were visualized using the ECL High Sensitivity Chemiluminescent Substrate (4 A BIOTECH, Suzhou, China). GAPDH (1:20,000, 60004–1-Ig), TRIB3 (1:500, 13300–1-AP), HRP-conjugated anti mouse IgG (1:5000, SA00001–1) and anti-rabbit IgG (1:5000, SA00001–2) were obtained from Proteintech (Wuhan, Hubei, China).
4
Western Blot Protein Detection Protocol
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Radioimmunoprecipitation assay lysis buffer (P0013, Beyotime) was added to collected tissues or cells to extract protein. The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, China). The protein samples were separated using SDS-PAGE and transferred to a 0.45-um PVDF membrane (Merck, Germany). Membranes were blocked by QuickBlock Western blocking solution (P0252, Beyotime, China) for 1 h and incubated with different kinds of primary antibodies (Table 1 ) at 4 °C overnight with rotation (the housekeeping proteins used in this study were GAPDH (whole protein/cytoplasmic protein) and Lamin B1 (nuclear protein)). TBST was used to wash off the unbound antibodies, and the membranes were incubated with the secondary antibodies of the corresponding species. secondary antibody was added, and the membrane was incubated for 1.5 h and washed again. Then, the membrane was covered with ECL working solution (1705062, Bio-Rad, USA) and incubated for 1–2 min. The membrane was placed in the imaging system for imaging, and Image Lab software was used for analysis of the gray value.
5
Detailed Western Blot Protocol
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Western blot was performed as previously described:51 (link) total protein was extracted from cell or tissue samples using RIPA buffer composed of 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate and complete protease cocktail (Roche) on ice. Samples were sonicated four times for 5 s on ice water at 15% power (Ningbo Scientific Biotechnology). Tissue lysates were centrifuged at 4 °C for 10 min at 14,000 g and supernatant was mixed with 4× LDS Sample Buffer (NP0007) and boiled for 10 min at 70 °C. The 10-μg protein samples were separated by 4–12% SDS–PAGE with MOPS-SDS running buffer and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were blocked with 5 QuickBlock™ Western Blocking Solution (P0252, Beyotime Biotechnology) for 1 h and then incubated with primary antibodies prepared using Western Primary Antibody Diluent (P0256, Beyotime Biotechnology) at 4 °C overnight. Membranes were incubated with HRP-conjugated secondary antibodies prepared using QuickBlock™ Western Secondary Antibody Diluent (P0258, Beyotime Biotechnology), and incubated for 1 h at room temperature. Immunoreactions were captured by X-ray films detecting chemiluminescence using an ECL kit (GE Healthcare). The full scan blots can be found in the Fig. S16 .
6
Western Blot Analysis of Protein Expression
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After treating the cells with PS-NPs at concentrations of 0, 10, 20, 50, and 100 μg/mL for 24 h, the HTR cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing proteinase and phosphatase inhibitors (Beyotime Biotechnology, China). Protein concentration was measured using a BCA reagent kit and proteins were denatured by adding SDS and PBS and heating at 100 °C for 5 min. After gel electrophoresis, the proteins were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. QuickBlock Western Blocking Solution (Beyotime Biotechnology, China) was used for blocking for 20 min. After incubation with the primary antibody at 4 °C overnight, the membranes were washed with TBST (Servicebio, Wuhan, China), and then subjected to incubation with the secondary antibody for 1 h before being washed again with TBST. Finally, the PVDF membranes were imaged using a Bio-Rad ChemiDocXRS+ (Bio-Rad, Hercules, CA, USA). Protein band grayscale values on the images were calculated using Image J.
7
Protein Extraction and Western Blotting
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Total protein was extracted from the intramuscular adipocytes with a WB/IP lysis buffer supplemented with 1% PMSF (Beyotime, Shanghai, China). According to molecular weight, 15 μg of total protein was separated by polyacrylamide gel electrophoresis. The proteins on the PVDF membrane were blocked for 30 min with the Quick Block Western Blocking Solution (Beyotime, Shanghai, China). Subsequently, the PVDF membranes were incubated with primary antibodies and secondary antibodies for 12 h and 2 h at 4 ℃ and RT, respectively. Finally, a DocTMXR system (Bio-Rad, Hercules, California, USA) was utilized to expose the protein bands. The protein on the PVDF membranes was flushed with a 1 × TBST buffer in the above steps. Antibody information is provided in Additional file 3 .
8
Western Blot Analysis of Intervertebral Disc Proteins
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NP tissue of all caudal IVDs was obtained for western blot under aseptic conditions after exposing caudal IVDs according to previously reported methods.
32 (link) Radioimmunoprecipitation assay lysis buffer (P0013, Beyotime, China) was added to collected tissues to extract protein. The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, China). Electrophoresis and semidry transfer electrophoresis were performed. Equal amounts of total protein (20 μg) of each sample were separated on 5%–12% gradient SDS‐PAGE gel (P0012AC, Beyotime) and transferred to a 0.45‐um PVDF membrane (Merck, Germany). After the membrane was blocked by QuickBlock Western blocking solution (P0252, Beyotime, China) for 1 h, and subsequently incubated with different kinds of primary antibodies (Table2 ) at 4°C overnight with rotation (the housekeeping proteins used in this study were GAPDH). TBST was used to wash off the unbound antibodies, and then the membranes were incubated with the secondary antibodies of the corresponding species for 1.5 hours and washed again. Then, the membrane was covered with ECL working solution (1705062, Bio‐Rad, USA) and incubated for 1–2 min. The membrane was placed in the imaging system for imaging, and Image Lab software was used for analysis of the gray value.
32 (link) Radioimmunoprecipitation assay lysis buffer (P0013, Beyotime, China) was added to collected tissues to extract protein. The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, China). Electrophoresis and semidry transfer electrophoresis were performed. Equal amounts of total protein (20 μg) of each sample were separated on 5%–12% gradient SDS‐PAGE gel (P0012AC, Beyotime) and transferred to a 0.45‐um PVDF membrane (Merck, Germany). After the membrane was blocked by QuickBlock Western blocking solution (P0252, Beyotime, China) for 1 h, and subsequently incubated with different kinds of primary antibodies (Table
9
Osteogenic Differentiation Protocol
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After the cells were cultured for five days, the protein was collected by RIPA containing protease inhibitors. After the protein concentration was measured by the kit, a quarter volume of the loading buffer (Servicebio, G2013) was added, denaturing at 95°C for 10 min. The protein samples were resolved by SDS-PAGE (4–20% gels) and then transferred to PVDF membranes using a protein transfer instrument (eBlotL1, GENSCRIPT). After 15 min of QuickBlock™ Western blocking solution (Beyotime, P0252) at room temperature, the membrane was incubated overnight with the primary antibody at 4°C for RUNX2(CST, 8486s), ALP (Zenbio,220,678), and GAPDH (Proteintech,60004-1-I). qRT-PCR assay using Cell Total RNA isolation kit (FOREGENE, RE-O3113) was utilized to extract mRNA. HiScript III RT SuperMix (Vazyme, R323-01) was used to perform reverse transcription of mRNA. Then, the reverse transcription product was used as a template to perform qRT-PCR on a StepOnePlus thermal cycler (Applied Biosystems, Foster City, CA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) to analyze the difference in gene expression. The primers used were designed at the NCBI and synthesized by Tsingke Biological technology.
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