Cells lysates were prepared in protein extraction buffers composed of radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Jiangsu, China), 10% phosphatase inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) and 1% phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Jiangsu, China) for 30 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore Ltd, Tullareen Carrigtwohill, Co, Cork IRL Rev.) in a Mini Trans-Blot Electrophoretic Transfer System (BIO-RAD, Hercules, CA, USA). After that, the membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated overnight with primary antibodies at 4°C. The following primary antibodies were used: mTOR (2983, Cell Signaling Technology, Danvers, MA, USA) and p-mTOR (2971, Cell Signaling Technology, Danvers, MA, USA), S6K1(14485, Proteintech Group, Inc., IL, USA) and p-S6k1 (ab32525, abcam, USA), and GAPDH (ab-p-r001,Goodhere Biotechnology CO., LTD, Hangzhou, China). The dilution ratio of antibodies above were 1:1000.
Mini trans blot electrophoretic transfer system
Manufactured by Bio-Rad
Sourced in United States
The Mini Trans-Blot Electrophoretic Transfer System is a lab equipment designed for the transfer of proteins from polyacrylamide gels to membranes. It facilitates the process of Western blotting, which is a widely used technique in molecular biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean 3 electrophoresis system, by Bio-Rad (3 mentions)
Immobilon p, by Merck Group (2 mentions)
Standard bicinchoninic acid assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Ab32525, by Abcam (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Hrp conjugated goat anti rabbit or anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Complete mini edta free protease inhibitor, by Roche (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Goat anti mouse igg conjugated with horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Mouse monoclonal anti c myc antibody, by Santa Cruz Biotechnology (1 mentions)
9 protocols using mini trans blot electrophoretic transfer system
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of P2X7R in Rat Retinas
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Western blot analysis was conducted as previously described with some modifications [3 (link)]. After washed with PBS solution, rat retinas or cells were lysed in lysis buffer (containing 1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined by the BCA method (Pierce). Protein samples (1.0 g/L, 15 L) were subjected to 10% SDS-PAGE using a Mini-Protean 3 electrophoresis system (Bio-Rad) and electrotransferred to polyvinylidene fluoride membranes using a Mini TransBlot electrophoretic transfer system (Bio-Rad). The membranes were blocked with 5% skimmed milk at room temperature for 1 h and then incubated with rabbit antibody against-P2X7R (1 : 1000, Abcam). The blots were washed with TBST and incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (1 : 4000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature and visualized with enhanced chemifluorescent reagent ECL (Thermo Scientific, Rockford, IL, USA) and exposed to X-ray film in the dark. The experiments were performed in triplicate, and the protein bands were quantitatively analyzed with Image J Analysis software.
3
Western Blot Analysis of Protein Expression
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Cells were washed with ice cold PBS and lysed by RIPA buffer (Sigma) containing complete Mini EDTA-free protease inhibitor (Roche Molecular Biochemicals, Indianapolis, IN). Protein concentration in each cell lysate was then measured using Bicinchoninic Acid (BCA) assay kit (Thermo Scientific, Rockford, USA), following the manufacturer’s procedure. Loading dye and β-mercaptoethanol (Sigma) was added to the samples and heated at 95°C for 10 min, followed by centrifugation at 13,000xg for 10 min. In each lane of SDS-PAGE gel (12%), 25μg of protein sample was loaded, stacked at 60V for 30 min and separated at 110V for 1h. The resolved proteins were then transferred to PVDF membrane (Bio-Rad Laboratories) at 90V and 4°C overnight using a Bio-Rad Mini Trans-Blot Electrophoretic Transfer System. Membranes were blocked for 1h at room temperature with 5% non-fat milk and incubated with different primary antibodies at 4°C overnight. Membranes were washed and incubated with horseradish peroxidase (HRP)-labelled secondary antibodies for 2h at RT. Immuno-reactive bands were then visualized using Immobilon Western Chemiluminescet HRP Substrate (Cat#WBKLS0100 Millipore Corporation, Billerica, MA,USA). Imaging was performed on a Biorad chemidoc platform and level of intensities were analyzed using Bio-Rad Quantity One software.
4
Induction and Measurement of Fusion Proteins
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G418-resistant transformants were grown overnight in buffered methanol-complex medium at 30°C and shaking at 280 r/min in 50 mL centrifuge tubes until the absorbance at 600 nm was 2–6. The cells were recovered by centrifugation and resuspended in buffered methanol-complex medium to 1.0 of an absorbance for induction, and grown again at 30°C, 280 r/min in 250 mL glass culture tubes. Fresh methanol was added to a total of 1% to maintain induction every 24 hours. Fusion proteins were measured at 24, 48, 72, 96, and 132 hours post-induction in 1 mL media by centrifugation. After 132 hours, the supernatants were concentrated 25 times by lyophilization. Samples of the supernatants were analyzed by electrophoresis on NEXT GEL™ 10% (Amresco, Solon, OH, USA) followed by Coomassie brilliant blue (Sigma, St. Louis, MO, USA) staining and western blot analysis. Western blot analysis was achieved using a wet blotting method with the MINI-TRANS-BLOT electrophoretic transfer system (Bio-RAD, Hercules, CA, USA), with NEXT GEL™ electrophoretic buffer (Amresco) at 90 V for 2 hours. The protein samples were detected using a mouse anti-c-myc monoclonal antibody (1 μg/mL; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-mouse IgG conjugated with horseradish peroxidase (0.08 μg/mL; Jackson ImmunoResearch, West Grove, PA, USA). The horseradish peroxidase activity was visualized with DAB.
5
Western Blot Protein Extraction Protocol
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Cells pellets were washed twice with ice-cold PBS, resuspended in laemmli sample buffer 1× + 100 mM Dithiothreitol (DTT) and sonicated with five 1 min on/off cycles with Bioruptor (Diagenode). Protein electrophoresis was performed on polyacrylamide gels (BioRad) and proteins transferred to nitrocellulose membranes (GE Healthcare, Amersham Hybond ECL) with a Mini Trans-Blot Electrophoretic Transfer System (Biorad). Lysis buffer was complemented with Protease Inhibitor Cocktail Tablets (Roche).
6
Cell Lysis and Western Blotting
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Cells were collected by trypsinization. After washing with PBS, the cells were lysed in 10 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% deoxycholate supplemented with 1 mM PMSF (Sigma) and protease inhibitor cocktail (Roche) for 30 min cell. Lysates were centrifuged and the supernatant was boiled with Laemmli buffer. SDS-PAGE was performed as previously described [58 (link)]. Transfer to membrane was done using a BioRad Mini-Transblot Electrophoretic Transfer System. The membranes were subsequently blocked in 5% non-fat milk in TBS-T. The following primary antibodies were used: anti-C-Nap1 (BD Biosciences), anti-GAPDH (Cell Signalling Technology), anti-α-tubulin (Sigma, T-9026); with appropriate secondary antibodies: donkey HRP-coupled anti-mouse and donkey HRP-coupled anti- rabbit antibodies (from Jackson laboratories).
7
Western Blot Analysis of C-Nap1 and Lamin B1
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Proteins were separated on 6% SDS–PAGE gel and transferred to a PVDF membrane using a Bio‐Rad Mini‐Transblot Electrophoretic Transfer System. Membranes were blocked with 5% milk in TBST and probed with primary antibodies C‐Nap1 (rabbit, 1:300, 14498‐1‐AP, Proteintech), Lamin B1 (rabbit, 1:1,000, ab16048 Abcam) diluted in 3% BSA in TBST. The membrane was subsequently blocked with HRP‐conjugated secondary antibodies, anti‐rabbit IgG (H + L) (donkey, 1:3,000, 711‐035‐152, Jackson) diluted in 5% milk TBST at RT for 1 h.
8
Retinal Protein Extraction and Western Blotting
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Western blotting analyses were performed as described previously [18] [19] (link)[20] (link). For whole cell protein extraction, retinas were homogenized in the RIPA lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). The extraction of retinal plasma membrane protein was followed the instructions of a commercial plasma membrane protein extraction kit (Bio Vision, Milpitas, CA, USA). The protein concentrations were determined by standard bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL, USA). According to the molecular weights of the target proteins, the protein samples were separated by 8%, 10% or 15% of twelve alkyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) respectively, and then transferred from the gels onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA) using Mini-PROTEAN 3 Electrophoresis System and Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad, Hercules, CA, USA). The antibodies used in this study were listed in the Supplementary Table 1. Digital imager (FluorChem E System, ProteinSimple, San Jose, CA, USA) was used for imaging, and AlphaView SA (version 3.4, ProteinSimple) was used for analysis.
9
Retinal Protein Extraction and Western Blotting
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Western blotting analyses were performed as described previously [18] [19] (link)[20] (link). For whole cell protein extraction, retinas were homogenized in the RIPA lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). The extraction of retinal plasma membrane protein was followed the instructions of a commercial plasma membrane protein extraction kit (Bio Vision, Milpitas, CA, USA). The protein concentrations were determined by standard bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL, USA). According to the molecular weights of the target proteins, the protein samples were separated by 8%, 10% or 15% of twelve alkyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) respectively, and then transferred from the gels onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA) using Mini-PROTEAN 3 Electrophoresis System and Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad, Hercules, CA, USA). The antibodies used in this study were listed in the Supplementary Table 1. Digital imager (FluorChem E System, ProteinSimple, San Jose, CA, USA) was used for imaging, and AlphaView SA (version 3.4, ProteinSimple) was used for analysis.
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