Preadipocytes were washed with PBS, lysed using RIPA buffer (Beyotime, Shanghai, China) on ice, and centrifuged at 10,000 ×g at 4°C. The whole protein concentration for each sample was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China). All protein samples and SDS-PAGE Sample Loading Buffer (Beyotime, Shanghai, China) were mixed at a ratio of 4 to 1 and boiled for 5 min. Next, all protein samples were diluted to the same concentration. Protein bands were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% Blocking Buffer (Beyotime, Shanghai, China) for 1 h. Next, primary antibodies directed against SLC25A1, DLST, or actin (Cell Signaling Technology, CST, USA) were incubated on the membranes at 4°C overnight. The membranes were then treated with HRP-conjugated secondary antibodies (Cell Signaling Technology, CST, USA) at room temperature for 2 h. Signals were detected using the enhanced chemiluminescence system reagents (Amersham Pharmacia Biotech, USA). All experiments were repeated in triplicate.
Blocking buffer
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
Blocking buffer is a laboratory reagent used to prevent non-specific binding during immunoassays and other protein detection techniques. It helps to minimize background signals and improve the specificity of the target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (25 mentions)
Bca protein assay kit, by Beyotime (18 mentions)
Ripa lysis buffer, by Beyotime (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (11 mentions)
Bca kit, by Thermo Fisher Scientific (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Chemidoc xrs system, by Bio-Rad (6 mentions)
Beyoecl plus, by Beyotime (6 mentions)
Lysis buffer, by Beyotime (6 mentions)
Triton x 100, by Beyotime (6 mentions)
Ripa buffer, by Beyotime (5 mentions)
Pvdf membrane, by Beyotime (5 mentions)
Total protein extraction kit, by Beyotime (4 mentions)
Gapdh, by Abcam (4 mentions)
Bca kit, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Enhanced chemiluminescence reagent, by Merck Group (3 mentions)
Ab205718, by Abcam (3 mentions)
175 protocols using blocking buffer
1
Western Blot Analysis of Preadipocyte Proteins
2
Quantitative Protein Analysis in ATDC5 Cells
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Total protein in ATDC5 cells after relevant transfection and treatment were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). The purity of proteins was quantified by the BCA™ Protein Assay Kit (Pierce, Appleton, WI). Western blot system was established using a Bio-Rad Bis-Tris Gel system (Bio-Rad Laboratories, Hercules, CA). Proteins in equal concentration were electrophoresed in PAGE-SDS and transferred onto PDVF membranes (Millipore, Bedford, MA). After blocking in the 5% blocking buffer (Beyotime), the membranes were incubated with primary antibodies which were prepared in 5% blocking buffer at a dilution of 1:1, 000. After incubation with primary antibodies at 4°C overnight, the PDVF membranes were incubated with secondary antibody for 1 h at room temperature. Signals of the bands were captured using Bio-Rad ChemiDoc TM XRS system (Bio-Rad). The intensity of the bands was quantified using Image Lab™ Software (Bio-Rad).
3
Western Blot Analysis of Cell Signaling Proteins
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The cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA, 0.1 mM PMSF, and 2 mg/mL aprotinin. Protein concentration was measured using the BCA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Then, the proteins (50 µg/lane) were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime Biotech, Shanghai, China) and transferred onto nitrocellulose membranes. The membranes were blocked with 3% blocking buffer (Beyotime) for 1 hour at room temperature and cultured with the primary antibodies CNNM1 (1:1,000, ab122648, Abcam), BAX (1:1,000, ab32503, Abcam), bate-actin (1:1,000, ab6276, Abcam), and BCL2 (1:1,000, ab32124, Abcam) overnight at 4 ℃. Subsequently, the membranes were cultured with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000, ab205718, Abcam) for 2 hours. Next, the membranes were visualized using an enhanced chemiluminescence reagent (Millipore). Protein blotting was analyzed using the Image Pro Plus 6.0 software (National Institutes of Health), with bate-actin as the internal reference. The experiment was repeated three times.
4
Western Blot Analysis of TNFAIP3 and CD20
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Patient focus or mouse tumor tissue homogenate or cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA, 0.1 mM PMSF, and 2 mg/ml aprotinin. Protein concentration was measured using the BCA kit (Beijing Solarbio Science & Technology Co., Ltd.). Then, the proteins (50 µg/lane) were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime Biotech) and transferred onto nitrocellulose membranes. The membranes were blocked with 3% blocking buffer (Beyotime) for 1 h at room temperature and cultured with the primary antibodies TNFAIP3 (1:1,000, ab92324, Abcam) and CD20 (1:1,000, Abcam) overnight at 4°C. Subsequently, the membranes were cultured with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000, ab205718, Abcam) for 2 h. Next, the membranes were visualized using an enhanced chemiluminescence reagent (Millipore). Protein blotting was analyzed using the Image Pro Plus 6.0 software (National Institutes of Health), with β-actin as the internal reference. The experiment was repeated three times.
5
Western Blot Analysis of Myogenic Markers
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The antibodies used were MyoG (ab124800, Abcam), MyoD (ab133627, Abcam), p-ERK1/2 (9101, Cell Signaling Technology), GAPDH (ab8245, Abcam), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208; Beyotime). Total protein from cultured MuSCs was extracted using the Total Protein Extraction Kit (Beyotime) and quantified using the BCA Protein Quantitation Kit (Beyotime) according to the manufacturer’s instructions. Briefly, proteins (~20 μg per sample) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, Bedford, MA, USA), and blocked with blocking buffer (Beyotime) for 1 h at room temperature. The membranes were sequentially incubated with primary anti-mouse MyoG (1:1000), MyoD (1:1000) and p-ERK1/2 (1:1000), washed three times with PBST (0.1% Tween 20 in PBS), and incubated with the secondary antibody conjugated with HRP (1:4000) for 90 min. The membranes were washed three with PBST. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (BeyoECL Plus, Beyotime) and the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA). GAPDH (1:2000) served as a loading control.
6
Exosome Marker Detection Protocol
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The protein concentration of cell lysis was measured. SDS PAGE gel (P0690, Beyotime, China) and PVDF membranes (IPVH00010, Millipore, USA) were needed. Blocking was conducted with Blocking Buffer (P0231, Beyotime). Antibodies were listed in Table 3 . The enhanced chemiluminescence reaction (Kibbutz Beit Haemek, Israel) and Alpha Innotech Flour ChemFC2 imaging system (Alpha Innotech) was used for detection.
Antibodies Used for Western Blot Assay
| Antibody | Dilution | Product ID | Function | Source |
|---|---|---|---|---|
| CD63 | 1/1000 | ab59479 | Exosomes marker | Abcam |
| TSG101 | 1/1000 | ab125011 | Exosomes marker | Abcam |
| Calnexin | 1/20000 | ab92573 | ER membrane marker | Abcam |
| GAPDH | 1/500 | ab8245 | loading control antibody | Abcam |
| ITM2B | 1/1000 | ab129282 | protease inhibitor | Abcam |
| β-actin | 1/1000 | ab8226 | loading control antibody | Abcam |
7
Nuclear Factor-kappa B Localization
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Cells were seeded and transfected with NC or ZHX2 siRNA in 6‐well plates. After 48 hour, cells were collected and fixed with 4% paraformaldehyde, then permeabilized with Triton X‐100, and blocked with blocking buffer (Beyotime). Samples were incubated with the primary antibody anti‐P65 (NF‐κB, CST) and fluorescein‐labeled secondary antibody (CST), and then DAPI (Beyotime) was used to stain the nucleus.
8
Protein Expression Analysis of Colon and BMSC
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Total colon tissues and BMSCs cells were extracted by RIPA lysis buffer with 0.1% PMSF (Beyotime, China). Protein quantification was performed with the BCA assay kit (Beyotime). The homogenized protein samples were fractionated by 4%–20% pre‐cast gel (GenScript) at 120 V for 1.5 h, and transferred onto nitrocellulose filter (NC) membranes (Bio‐Rad Laboratories,USA) at 110–120 V for 1 h. The membranes were blocked in blocking buffer (Beyotime, China) for 15–30 min at room temperature, followed by an overnight incubation at 4°C with primary antibodies (Anti‐Occludin, #91131, 1:1000; Anti‐E‐cadherin, #14472, 1:1000; Anti‐LC3B, ab192890, 1:1000; Anti‐Caspase‐3, ab32351, 1:1000), and respective fluorescent secondary antibodies (Goat anti‐Mouse IgG H&L IRDye 680RD, ab216776, 1:5000; Goat anti‐Rabbit IgG H&L IRDye 800CW, ab216773, 1:5000) at 37°C for 1–2 h. Finally, the immunoreactive bands were visualized using Odyssey Clx fluorescence scanning system (LI‐COR Biosciences, USA). Equal protein loading was normalized with Anti‐β‐actin antibody.
9
Western Blot Analysis of Autophagy and Apoptosis Markers
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Proteins were extracted from the isolated tissues containing Kölliker’s organs of 20 animals. The proteins were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer (Beyotime, Shanghai, China) at room temperature for 1 h and then incubated with primary antibodies against β-actin (Beyotime; AA128, 1:1000), MAP LC3II (Santa Cruz Biotechnology; sc-271625, 1:1000), P62 (Abcam; ab109012, 1:1000), Beclin1 (Abcam; ab62557, 1:1000), caspase-3 (CST; #9662, 1:1000), cleave-caspase3 (CST; #9664, 1:1000), Bcl-2 (Wanleibio, Shanghai, China; WL01556, 1:2000) and GAPDH (PTG, 60004-1-lg, 1:45000) at 4°C overnight. After three times of washing with PBS-0.01% Tween 20 (PBS-T), the membranes were incubated with a secondary antibody, anti-rabbit IgG or antimouse IgG (Beyotime; 1:1000), for 1 h at 37°C. After washing the membranes, and adding freshly prepared chemiluminescence solution (Millipore; A:B=1:1), the immunoreactive bands were imaged and analyzed under the Bio-Rad ChemiDoc XRS+ (Bio-Rad Co., Hercules, CA, USA) instructions.
10
Mincle Protein Detection in Murine Corneas and Cells
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Mice corneas (n = 5/group/time) for 3 and 5 days p.i. and RAW264.7 cells after stimulation of inactivated hyphae or TDB for 24 h were collected for detection of Mincle protein levels, which were fully lysed in RIPA solution (Solarbio, Beijing, China) with 1% PMSF and Phosphatase inhibitor. Concentrations of protein were then determined by a BCA Assay Kit (Elabscience). Separated proteins in PVDF (polyvinylidene fluoride) membranes (Solarbio) were transferred from SDS-PAGE glue. After 2 h’s blocking with a blocking buffer (Beyotime Biotechnology, Shanghai, China), the PVDF membranes containing proteins were incubated with goat anti-mouse Mincle (1:500, Santa Cruz, CA, United States) antibody or goat anti-rabbit β-actin (1:2000, Elabscience) antibody overnight at 4°C and with the corresponding secondary antibody (1:2000, Elabscience) for 2 h at 28°C. Chemiluminescence (ECL; Thermo Fisher Scientific, United States) was used to inspect the blots.
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