Tissue samples in each group were obtained from the rat wound area and stored at −80°C before Western blotting. Samples were ground in liquid nitrogen and subsequently homogenized in the RIPA lysis buffer containing protease inhibitor cocktail. The extracts above were collected after centrifugation. Protein concentrations were quantified using the BCA assay. The protein (40 ug) was separated by 7.5–12.5% polyacrylamide gel and then transferred onto PVDF membranes (BioRad Hercules, CA, USA). After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C overnight with the following primary antibodies: anti-matrix metallopeptidase2 (MMP-2) (1:1,000), anti-fibronectin (1:1,000), anti-laminin (1:1,000), anti-LC3 (1:1,000), anti-VEGF (1:1,000), anti-CD31 (1:1,000), mouse monoclonal anti-sQSTM1/p62 (1:1,000), PI3K (1:1,000), p-PI3K (1:1,000, 4257), AKT (1:1,000), p-AKT (1:1,000), mTOR (1:1,000), p-mTOR (1:1,000), and GAPDH (1:000), respectively. After 16 h, the membranes were incubated with goat anti-rabbit (1:1,000, BS13278, Bioworld) or goat anti-mouse (1:1,000, BS13278, Bioworld) secondary antibodies for 2 h at room temperature. The immunoreactive proteins were visualized using a Chemi DocXRS + Imaging System (BioRad). Finally, band intensity was quantified by Image J.
Bs13278
Manufactured by Bioworld Technology
Sourced in United States, China
The BS13278 is a laboratory centrifuge designed for high-speed separation of biological samples. It features a maximum speed of 13,000 RPM and a maximum relative centrifugal force of 20,000 × g. The centrifuge accommodates a variety of rotor options to suit different sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Bs12478, by Bioworld Technology (4 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Proteinase inhibitor, by Roche (2 mentions)
Sa00001 3, by Proteintech (2 mentions)
Sa00001 1, by Proteintech (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab109021, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab179483, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Ab188224, by Abcam (1 mentions)
P gsk 3β, by Bioworld Technology (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Gsk3β, by Bioworld Technology (1 mentions)
Ab108319, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Applygen (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Immobilon p, by Merck Group (1 mentions)
22 protocols using bs13278
1
Protein Expression Analysis of Wound Healing
2
Protein Extraction and Antibody Analysis in Mouse Crypts
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After isolation of crypts in C57BL/6 mice, total proteins were extracted from those cells using RIPA lysis buffer (1% PMSF) (Solarbio Science & Technology, China). The primary antibodies included anti-EGFR (1:1,000; catalog no. Ab52894, Abcam, UK), anti-NOS2 (1:1,000; catalog no. Ab178945, Abcam, UK), anti-HIF-1α (1:500; catalog no. Ab179483, Abcam, UK), anti-FIH (1:1,000; catalog no. Ab178945, Abcam, UK), anti-Cdkn1a (1:1,000; catalog no. Ab188224, Abcam, UK), and anti-HPRT (1:10,000; catalog no. Ab109021, Abcam, UK). Goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) (1:10,000; catalog no. BS13278, Bioworld Technology) was used as a secondary antibody.
3
Western Blot Analysis of BDNF Signaling
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At the end of experiment, all rats were sacrificed with 10% chloral hydrate solution (3.5 ml/kg; i.p.). Hippocampus and prefrontal cortex tissues were collected and lysed with radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China), schizolysised for 20 min on ice and centrifuged at 12,000 × g for 10 min at 4°C. Protein samples were heated at 95°C for 8 min, separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membrane were blocked for 2 h in TBST (25 mM Tris, 140 mM NaCl, 27 mM KCl and 0.02% Tween 20) containing 5% bovine serum albumin and incubated with primary antibodies specific for BDNF (ab108319, 1:200)(Abcam, Cambridge, MA, USA), TrkB (BS1431, 1:500), ERK (AP0491, 1:500), pERK (BS4621, 1:500), Akt (BS1502, 1:500), PI3K (BS3678, 1:500), pGSK3β (BS4084, 1:500) and GSK3β (BS1402, 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA), pCREB (#9198, 1:1,000) and CREB (#9197, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C over night. Following three washes with TBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibodies (BS13278, 1:5,000; Bioworld Technology, Inc.), washed with TBST three times. Blots were developed using a electrochemiluminescence system (UVP LLC, Upland, CA, USA).
4
Western Blot Analysis of ATP7A Protein
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The Western Blot was performed as previously described (27 (link)). In brief, total protein of cells was extracted with RIPA buffer (Beyotime, China) supplemented with protease inhibitor, phenylmethylsulfonyl fluoride (PMSF, Beyotime, China) at 4°C, 14000 rpm for 20 min. BCA assay (Fudebio, China) was used for protein quantification. The protein was isolated by electrophoresis using 10% SDS-polyacrylamide gel (PAGE) and then transferred to the PVDF membrane (Immobilon P, Millipore, USA). 5% fat-free milk (Beyotime, China) in Tris-buffered saline with Tween-20 (TBST) was used to block nonspecific binding sites at room temperature for 1h. Then, the membranes were immunoblotted with primary antibody overnight at 4°C: anti-ATP7A (1:1000, D161470, Sangon Biotech, China) and anti-GAPDH antibody (1:1000, AP0066, Bioworld Technology, USA). After being washed with TBST 3 times, blots were incubated with secondary peroxidase-conjugated antibodies, the goat anti-rabbit secondary antibody (1:5000, BS13278, Bioworld Technology, USA) for 1 h at room temperature. The membranes were performed with an enhanced chemiluminescence system and scanned by the Syngene Imaging System (Frederick, USA).s
5
Protein Expression Analysis of Cardiomyocytes
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NRVMs were washed with cold PBS and lysed with 1
× SDS-PAGE sample buffer (45 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.01% bromophenol blue, and 0.05 mM DTT) supplemented with 1 mM PMSF and a protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were boiled at 99°C for 10 min, separated by 12% SDS-PAGE, and then transferred onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at a room temperature, followed by overnight incubation at 4°C with the appropriate primary antibodies against C/EBP homologous protein (CHOP) (1:500, sc-7351; Santa Cruz Biotechnology, Dallas, USA), ubiquitin (1:500, sc-8017; Santa Cruz Biotechnology), GRP78 (1:500, BS6479; Bioworld Technology, Shanghai, China), eukaryotic initiation factor 2α (eIF2α) (1:500, BS3651; Bioworld Technology), and phosphorylated eIF2α at Ser51 (p-eIF2α) (1:500, BS4787; Bioworld Technology). The membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (1:10,000, BS13278, BS12478; Bioworld Technology). Finally, the protein blots were visualized using an enhanced chemiluminescence kit (Bio-Rad, Hercules, USA) with the Fluor Chem E System (Cell Biosciences, Santa Clara, USA). To promote ER stress in NRVMs, we used thapsigargin (1.0 μM, T9033; Sigma-Aldrich Chemical) as a positive control.
6
Western Blot Analysis of KLF12, Nur77, and GAPDH
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Proteins were extracted as described previously [17 (link)]. The protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts (25 μg) of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed by incubating the membranes with primary antibodies against KLF12 (1:2000; sc-84347, rabbit Polyclonal Antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nur77 (1:1000; 3960, rabbit Monoclonal Antibody, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:10000; AP0063, GAPDH polyclonal antibody, Bioworld Technology, MN, USA), followed by incubation with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000; BS13278, Bioworld Technology, St. Louis Park, MN, USA) and Flag-HRP (1:5000; A8592, Sigma, St. Louis, MO, USA). Detection was performed using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad, Hercules, CA, USA) software.
7
Quantifying Androgen Receptor Expression in Pelodiscus sinensis
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The samples were homogenized in ice-cold RIPA buffer (25 mM Tris/HCl (pH 7.6), 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet-P40, 0.1% SDS, and 0.05 mM PMSF). The protein concentration was quantified using a BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Equal amounts of protein (40 μg/lane) were subjected to 8% SDS-PAGE and subsequently transferred to polyvinylidene di-fluoride (PVDF) (Millipore, Bedford, MA) membranes. After blocking in 5% fat-free dry milk, the membranes were incubated overnight at 4 °C with an anti-AR antibody (ab198394, Abcam Inc., Cambridge, MA, USA) diluted 1:1000. After washing, the membrane was incubated with peroxidase-linked goat anti-rabbit IgG (1:5000, BS13278, Bioworld Technology Inc., Louis Park, MN) for 2 h. Bound antibodies were detected using an ECL detection system (Vazyme Biotech, China). The immunoreactive bands were quantified using Quantity One software (Bio-Rad Laboratories). The validity of AR antibody in P. sinensis has been detected by negative and positive control analysis.
8
Western Blot Analysis of SLC22A3
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Western blot assays were performed as standard procedures. The primary antibodies used were monoclonal rabbit anti‐SLC22A3 (1:1,000; ab151698; Abcam) and rabbit anti‐b‐actin (1:1,000; 13E5; Cell Signaling Technology). The secondary antibody was anti‐rabbit HRP (1:1,000; BS13278, Bioworld Technology). The immune complexes were detected by enhanced chemiluminescence (Cell Signaling Technology).
9
Collagen and Actin Protein Analysis
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Cells were lysed in RIPA lysis buffer (Beyotime, China), and the total protein concentrations were detected using the BCA Protein Assay Kit (TaKaRa, Japan). The proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes. All membranes were blocked with 5% BSA. The membranes were incubated with a rabbit polyclonal anti-human collagen type I antibody (ab34710; Abcam, dilution 1 : 4000), a rabbit polyclonal anti-human collagen type III antibody (ab7778; Abcam, dilution 1 : 4000), an anti-alpha muscle actin antibody (ab5694; Abcam, dilution 1 : 1000), or β-actin (BS13278, Bioworld, dilution 1 : 1500) at 4°C overnight. Subsequently, each membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h. Finally, we used the enhanced chemiluminescence kit to observe protein bands in a ChemiDoc XRS Plus c (Bio-Rad, USA).
10
Quantification of Protein Expression by Western Blot
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Total protein was extracted with RIPA lysis buffer (ABclonal, Wuhan, China), and quantified by bicinchoninic acid (BCA) assay. After sample preparation, protein samples (20 μg) were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membrane was incubated overnight at 4 C with the following primary antibodies: anti-USP25 (diluted 1:1,000, A7975, ABclonal, Wuhan, China), anti-TRAF6 (diluted 1:500, D21G3, Cell Signaling Technology, Boston, United States) and anti-GAPDH (diluted 1:10,000, ab181602, Abcam, Cambridge, United Kingdom). The membrane was then incubated with secondary antibody (anti-rabbit, 1:10,000, BS13278, Bioworld, Minnesota, United States) for 2 h at room temperature. After washing three times in TBST, protein bands were visualized with enhanced chemiluminescence (ECL) reagent (ABclonal, Wuhan, China). The gray values of the protein bands were quantified with ImageJ software (ImageJ 1.53, NIH, United States). Samples with poor expression of GAPDH were excluded, and the results were statistically evaluated by Student’s t test. Differences with a p value <0.05 were considered statistically significant.
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