Total protein from chondrocytes were extracted, and protein concentration was determined using BCA Protein Assay Kit (Beyotime; Shang Hai, China). Equal amount of protein samples (30 μg) was separated by 10% or 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a BioTrace NT Nitrocellulose transfer membrane (Pall). The membranes were than blocked with 5% non‐fat milk in tris buffered saline tween buffer for 1 or 2 h, and then incubated with primary antibodies against NLRP3 (1:1000, ABclonal, A12694), ASC (1:1000, ABclonal, A16672), caspase‐1 (1:1000, ABclonal, A0964), GSDMD (1:2000, ABclonal, A10164), MMP‐13 (1:1000, Novus, OTI2D8), ADAMTS‐5 (1:1000, Gene Tex, GTX123657), Adenosine A3 receptor (A3AR, 1:1000, Absin, abs121848) and GAPDH (1: 1000, ABclonal, A19056) overnight at 4°C. After that, rabbit HRP‐conjugated secondary antibody (1:3000, ZSGB‐BIO, ZB2306) was added and incubated at room temperature for 2 h. Intensity values were analysed using the ImageJ software (n = 3).
Zb 2306
Manufactured by ZSGB-BIO
Sourced in China
The ZB-2306 is a centrifuge device designed for separating mixtures of different densities. It utilizes high-speed spinning to create a centrifugal force that separates components based on their relative densities. The core function of this equipment is to facilitate the separation and isolation of various substances within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
Af5393, by Affinity Biosciences (1 mentions)
Bsm 33036m, by Bioss Antibodies (1 mentions)
Ab21685, by Abcam (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab13537, by Abcam (1 mentions)
Ab203119, by Abcam (1 mentions)
Ab37152, by Abcam (1 mentions)
Enhanced chemiluminescence solution, by Beyotime (1 mentions)
Ab176882, by Abcam (1 mentions)
Anti e cadherin, by Bioworld Technology (1 mentions)
Anti vimentin, by Bioworld Technology (1 mentions)
Ab64693, by Abcam (1 mentions)
Bca assay, by BioTeke (1 mentions)
Ab52954, by Abcam (1 mentions)
Zb 5301, by ZSGB-BIO (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
14 protocols using zb 2306
1
Protein Extraction and Western Blot Analysis of Chondrocytes
2
Quantitative Protein Expression Analysis
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Differentially expressed proteins such as HMOX1, MP2K5, XRCC1 and A1AG were measured by Western blot. Human lung AEC-II cells (Lanmeng Biotechnology, Hebei, China) were harvested after the treatments. After lysis for 30 min on ice with Cell Protein Extraction Reagent, the total protein concentration was determined by BCA assay according to the manufacturer's instructions. Each sample containing approximately 50 μg of proteins was electrophoresed on 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature. Primary antibodies (DF6623, AF5393, AF0296, DF7667, Affinity Biosciences, OH, USA) were incubated at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (ZB-2306, ZB-2305, ZSGB-BIO, Beijing, China). The immunoreactive bands were detected with an enhanced chemiluminescent (ECL) reagent kit according to the manufacturer's protocol and a chemiluminescence detection system. The obtained images were analyzed by Image J software to analyze the gray value of the target bands for relative quantitative information analysis of the proteins.
3
Protein Expression Analysis in Liver Tissues
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Liver tissues or hepatocytes were lysed in a cold lysis buffer (Keygene) supplemented with phenylmethanesulfonyl fluoride (PMSF) at 4°C for 30 min. After separation by 12% sulfate‐polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane. The membrane was blocked at room temperature in phosphate‐buffered saline with Tween 20 buffer with 5% nonfat milk followed by incubation with primary antibody, including anti‐ERO1α (ab172954, Abcam), anti‐GRP78 (ab21685, Abcam), anti‐PERK (ab156919, Abcam), anti‐ATF6 (ab203119, Abcam), and anti‐XBP‐1 (ab37152, Abcam), anti‐DNMT1 (ab13537, Abcam), anti‐G9a (#3306, Cell Signaling Technology), anti‐H3K9me2 (ab176882, Abcam), anti‐H3 (ab1791, Abcam) at 4°C overnight. Blotting of β‐actin (bsm‐33036M, BIOSS) was used as a loading control. The membrane was washed three times and incubated with horseradish peroxidase‐conjugated secondary antibody (ZB‐2306, ZSGB‐BIO) for 2 h. After washing three times, the target proteins were detected using enhanced chemiluminescence solution (Beyotime Institute of Biotechnology), and the relative protein levels were determined by band densitometry using a gel imaging system (Bio‐Rad Laboratories, Inc.).
4
Immunofluorescence Analysis of HK-2 Cells
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HK-2 cell slides were fixed, penetrated, and closed. Afterward, the slides were mixed with anti-fibronectin (1 μg/ml, Abcam, ab64693, U.K.), anti-α-SMA (1 μg/ml, Abcam, ab64693, U.K.), anti-E-cadherin, (1:200, Bioworld Technology, Inc., U.S.A.), and anti-vimentin (1:200, Bioworld Technology, Inc., U.S.A.) and incubated overnight. Then, the slides were incubated with secondary antibody (1:200, ZB-2306, ZSGB-Bio, Beijing, China) for 1 h according to the manufacturer’s instructions and analyzed with a fluorescence microscope.
5
Protein Expression Analysis in Rat Small Intestine
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The entire small intestine samples (2 cm regions of the proximal, medial, and distal of the rat small intestine) were homogenized in lysis buffer with 1 mM phenylmethylsulfonyl fluoride and centrifuged at 15,000 × g for 15 min. The protein concentrations were measured by the BCA assay (BioTeke Corporation, Beijing, China). After denaturation, proteins were subjected to SDS-PAGE, blotted onto polyvinylidene difluoride membranes and blocked in Tris-buffered saline containing 0.1% Tween-20 with 5% nonfat milk for 2 h. Then, the membranes were incubated with antibody against TPH1 (1:500) (ab52954, Abcam), SERT (1:250) (ARG63804, Arigo) or glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (1:1000) (TA802519, Origen) overnight at 4°C. After overnight incubation, the membranes were washed in Tris-buffered saline containing 0.1% Tween-20 thrice and incubated with anti-rabbit IgG (1:10000) (ZB-5301, ZSGB-BIO, Beijing, China), anti-goat IgG (1:15000) (ZB-2306, ZSGB-BIO, Beijing, China), and anti-mouse IgG (1:10000) (ATA0011, ATgene Biotech, Chongqing, China) for 2 h each and then visualized by electrochemiluminescence detection (Fujifilm, Japan).
6
Immunoblotting of IAV-OX40L in MDCK and HepG2 Cells
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MDCK and HepG2 cells were seeded in 6-well plates. When the cell density reached 70%, the cells were inoculated with IAV-OX40L. After 24 h, cell precipitates were collected and proteins extraction was performed using RIPA buffer (Solarbio R0010, Beijing, China). Protein concentrations were then determined by the BCA Protein Assay kit according to the manufacturer’s instructions. Next, 10 µg of protein was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Nonspecific binding was blocked with 5% bovine serum albumin (BSA) blocking buffer (SolarbioSW3015). NP polyclonal antibody (1:3000 dilution, GeneTex, GTX636247) and OX40L monoclonal antibody (1:1500 dilution, CST, 14,991) were applied overnight at 4°C. The secondary antibody was HRR-conjugated goat anti-rabbit (1:1000 dilution, ZSGB-bio, ZB2306) followed by HRR-conjugated goat anti-mouse (1:10,000 diluent, Protentech, SA00001-1). An enhanced chemiluminescence (ECL) detection system was used for observation.
7
Western Blot Analysis of Protein Expression
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To obtain the protein samples, the cells were collected, washed with PBS, and lysed in the RIPA lysis buffer (P0013B, Beyotime) supplemented with phenylmethanesulfonyl fluoride (PMSF, ST505, Beyotime). The protein concentrations were determined by a BCA protein assay kit (P0011, Beyotime); then the lysate was mixed with a 5× loading buffer and heated for 10 min at 100°C. The protein samples were loaded onto 10% SDS-PAGE and transferred to the PVDF membranes (IPVH00010, Millipore). After being blocked with 5% skimmed milk in the TBST buffer for 1 h, the PVDF membranes were incubated with specific primary antibodies (rabbit anti-Arf6 antibody, PA1-093, Invitrogen; rabbit anti-Rab5a antibody, 2143T, cell signaling technology; mouse anti-CD147 antibody, orb251620, Biorbyt; goat anti-ACE2 antibody, AF933, R&D Systems; mouse anti-tubulin antibody, 66031-1-Ig, Proteintech) at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (peroxidase-conjugated rabbit anti-goat IgG (H + L), ZB-2306, ZSGB-Bio; goat anti-rabbit IgG (H + L) secondary antibody, HRP, 31460, Invitrogen; goat anti-mouse IgG (H + L) secondary antibody, HRP, 31430, Invitrogen) for 1 h at room temperature and the images were obtained and analyzed using the Image Lab software (BIO-RAD).
8
Cochlear Protein Expression Analysis
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Three dissected cochleas in each group were collected on ice, stored at −80°C, and lysed using a RIPA buffer (Biyotime, Shanghai, China). The concentration of the extracts was determined with the Micro BCA kit (Solarbio, Beijing, China). Protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, MA, United States), which were blocked with 5% nonfat dry milk in TBS with 0.1% Tween-20. The membranes were incubated with primary antibodies, namely rabbit anti-Nrf2 (16396-1-AP, 1:1000, Proteintech, Wuhan, China), anti-HO-1 (110701-1-AP, 1:1000, Proteintech, Wuhan, China), anti-caspase-3 (19677-1-AP, 1:500, Proteintech, Wuhan, China), anti-iNOS (18985-1-AP, 1:500, Proteintech, Wuhan, China) and anti-β-actin (WL01372, 1:1000, Wanleibio, Shenyang, China) overnight. After incubation with the secondary antibody (ZB-2306, 1:5000, ZSGB-BIO, Beijing, China), and extensive membrane washing, the blots of immune reactivity were illustrated through enhanced chemiluminescence (KF001, Affinity Biosciences, OH, United States). ImageJ (Bethesda, MD, United States) was used for densitometric comparisons. β-actin density measurements were used as loading controls.
9
Protein Extraction and Western Blot Analysis
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The total proteins were extracted using RIPA buffer supplemented with a proteinase inhibitor cocktail (Beyotime, Beijing, China). Utilizing the BCA assay kit (Beyotime, Beijing, China), the protein content was determined. Polyacrylamide gel electrophoresis (SDS-PAGE) with the addition of sodium dodecyl sulfate was employed to separate the proteins, followed by their transfer onto the polyvinylidene fluoride membranes made from PVDF. After an hour of room temperature blocking with blocking solution, the membranes were incubated for an entire night at 4°C with primary antibodies targeting HK2 (dilution 1:5000, Cat No. 66974-1-Ig, Proteintech, China) and β-Actin (dilution 1:5000, Cat No. 81115-1-RR, Proteintech, China). Following a wash, the membrane was left at room-temperature for an hour to be incubated with the secondary antibody (dilution 1:10000, ZB-2306, ZSGB-BIO, China). An improved chemiluminescent substrate was used to identify protein bands.
10
Hippocampal Protein Expression Analysis
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The rat hippocampus was collected immediately after the animals completed the behavioral tests. Proteins of the tissues were extracted and the concentrations determined by BCA Protein Assay Kit (CWBIO, Beijing, China). Each sample contained with 40 μg of protein was separated by 10 or 12% SDS-PAGE and then transferred to nitrocellulose membranes (Millipore, MA). The following primary antibodies were incubated with the PVDF membrane at 4°C overnight. PBR (1:7,000 dilution, ab92291; Abcam), ATG7 (1:7,000 dilution, ab133528; Abcam), ATG5 (1:7,000 dilution, ab108327; Abcam), LC3B (1:2,000 dilution, ab192890; Abcam), p62 (1:7,000 dilution, ab109012; Abcam) and β-actin (1:7,000 dilution, aa128; Beyotime Biotechnology). After that, rabbit anti-goat immunoglobulin G (IgG) (H + L) horseradish peroxidase (HRP) (1:5,000 dilution, ZB2306; ZSGB-BIO), goat anti-rabbit immunoglobulin G (IgG) (H+L) HRP (1:7,000 dilution, GAR007; MultiSciences) or goat anti-mouse IgG (H+L) HRP (1:7,000 dilution, GAM007; MultiSciences) were incubated with the membrane for 2 h at room temperature. Bands were visualized with enhanced chemiluminescence (ECL) detection reagents (CWBIO, Beijing, China) using an ECL reagent. The relative band intensity was measured by Image-Pro Plus software.
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