Cells were lysed in RIPA lysis buffer (Millipore) and protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). Samples were separated by SDS-PAGE and electroblotted to polyvinylidene fluoride (PVDF) membranes (GE Healthcare). In the case of SDC1 detection, cell membrane extract was prepared using Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific) according to the manufacturer's protocol, and blotted to cationic nylon membrane (Immobilon Ny; Millipore). Signals were visualized with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer's protocol. Chemiluminescence was quantitated using a Bio-Rad ChemiDocTM MP imaging system.
Immobilon ny
Manufactured by Merck Group
Sourced in United States, Germany
IMMOBILON-NY+ is a nylon-based transfer membrane designed for the immobilization of nucleic acids. It is a high-performance blotting membrane that provides consistent and efficient transfer of nucleic acids from agarose or polyacrylamide gels to a solid support for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bamhi, by New England Biolabs (3 mentions)
Hindiii, by New England Biolabs (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase labeled anti dig antibody, by Roche (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoctm mp imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Subcellular protein fractionation kit for cultured cells, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Cyclone plus storage phosphor system, by PerkinElmer (1 mentions)
Rediprime 2, by GE Healthcare (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Normal mouse igg, by Merck Group (1 mentions)
Emsa kit, by Promega (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Zyppy plasmid kit, by Zymo Research (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Sepharose 4b, by Cytiva (1 mentions)
20 protocols using immobilon ny
1
Quantitative Western Blot Analysis
2
Total RNA Isolation from Cyanobacteria
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Total RNA isolation was performed by vortexing cell pellets from 40 ml cultures in the presence of acid-washed baked glass beads (0.25–0.3 mm diameter) and phenol–chloroform. Isolated RNA was quantified with a NanoDrop 1000 (Thermo Scientific), separated by electrophoresis on 1.2% agarose denaturing formaldehyde gels, and blotted on to nylon membranes (Immobilon-NY+, Millipore) (Sambrook and Russell, 2001 ). Prehybridization, hybridization, and washing steps were performed following the manufacturer’s recommendations. Probes for northern blot hybridization were obtained by PCR with oligonucleotide pairs sll1815_1F/sll1815_1R for adk, sll1823_1F/sll1823_1R for purA, and sll1566_1F/sll1566_1R for ggpS (sequences listed in Supplementary Table S1 ) and random-prime labeled (Rediprime II, GE Healthcare) with α-[32P] dCTP. Hybridization signals were detected and analyzed with a Cyclone Plus Storage Phosphor system (Perkin-Elmer). The signal for rnpB was used for normalization.
3
Runx2 Binding Analysis by EMSA
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Nuclear extracts were prepared from Dox-treated C2C12/Runx2Dox cells according to the manufacturer’s instructions (Active Motif, Carlsbad, CA, USA). EMSA was performed using the EMSA kit (Promega). For supershift assays, an antibody against Flag-Runx2 (anti-Flag antibody) (Sigma) or normal mouse IgG (Sigma) was added to the reaction mixture and incubated 25 min before the addition of the labeled oligonucleotide. All DNA–protein complexes were resolved by electrophoresis on 5 % native polyacrylamide gels and transmembrane to immobilon-Ny + (Millipore).
4
Plasmid Characterization via Restriction Digest
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One fully confluent T75 flask was used for each time point. Plasmids were recovered using Zyppy plasmid Kit (Zymo Research, Cat # D4019). 10ug of plasmid DNA from each time point was digested with either Csp45I and NotI or Csp45I alone. Heat inactivated samples were run in 1% agarose-gel. Blotting onto Immobilon-Ny + (Millipore, Cat # INYC00010) was achieved using Trans-Blot SD semi-dry transfer cell (Biorad, Cat # 170–3957). EKONO hybridization buffer (Research Products International, Cat # 248800) was used for both pre-hybridization and hybridization. Hybridization probe was synthesized by random priming using random hexamers (Invitrogen, Cat # 51709) using whole plasmid DNA as template. Finally, the blot was exposed to X-ray films and developed using standard developer.
5
STAT Protein Purification and Detection
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The shrimp intestine was lysed, and STAT protein was purified using anti-STAT antibody- CNBr-activated Sepharose 4B (60 mg; Amersham Biosciences AB, Uppsala, Sweden). The digoxigenin-labeled probes (sense, 5'-GCGTAAGGTTTTCTTGGAATA-3'; antisense, 5'-TATTCCAAGAAAACCTTACGC-3') were synthetized and labeled by Sangon Company (China). Two micrograms of purified proteins was mixed with 3 μl of 5× binding buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 10 min and incubated with 20 fmol of digoxigenin-labeled probe for 20 min. In competition experiments, unlabeled probe was pre-incubated with the relevant proteins for 10 min before the Dig-labeled probe was added and incubated for 20 min at room temperature. The reaction solution was run on a 6% polyacrylamide/0.5× TBE gel at 80 V, and the samples were transferred onto a nylon membrane (IMMOBILON-NY+, Millipore, Milford, MA, USA). The membrane was first blocked with blocking buffer for 30 min and then incubated with anti-Dig phosphatase antibody (1:10000 in blocking solution; Roche, Germany) for 1h. The signal was visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride.
6
Double-Filter Method for DNA-Protein Binding
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We performed a double-filter method as previously described (21 (link)). A nitrocellulose membrane was incubated for 10 min in 0.4 M KOH at 4°C, washed with water and equilibrated in 20 mM Tris HCl pH 8, 8% glycerol; Immobilon-NY+ (Millipore) was equilibrated in the same buffer. Immobilon membrane was placed below the nitrocellulose membrane in a filter binding apparatus Minifold I (Whatman) where the upper membrane captures MAZ-GST with or without bound DNA and the lower membrane traps free DNA. Samples containing DNA and MAZ-GST, incubated at 4 °C for 30 min in binding buffer (see EMSA) added with KCl as specified in figure captions, were applied to filters under vacuum and washed with binding buffer. Filters were dried, exposed for autoradiography and quantified using an Image Scanner controlled by ImageQuant TL V2003.03 software (GE Healthcare).
7
Northern Blot Analysis of comEA and mreB
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Northern-blot analysis were performed as previously described39 (link). Briefly, three micrograms of total RNA in denaturing buffer were loaded per lane and run on denaturing formaldehyde 1.5% agarose gel in MOPS buffer. RNA were transferred to nylon membrane (IMMOBILON-NY+, Millipore corporation) by capillary transfer in 10X SSC buffer (Saline Sodium Citrate 1X: 150 mM NaCl + 15 mM sodium citrate) and then cross-linked to the membrane by UV irradiation. Membrane were hybridised at 42 °C with 5′-biotinylated oligonucleotide probes (5 nM) for either comEA (comEA2-NB) or mreB (mreB-NB) in ULTRAhyb Ultrasensitive Hybridisation Buffer (Ambion, Austin, TX). Following overnight hybridisation the membranes were washed twice in 2X SSC buffer containing 0.1% SDS at 65 °C according to the ULTRAhyb manufacturer instructions. Probed membranes were revealed using horseradish peroxidase-conjugated streptavidin and enhanced luminol substrate (Chemiluminescent Nucleic Acid Detection Module, Pierce, Rockford, IL). Luminescence signals were acquired using an imaging workstation equipped with a charge-coupled device camera (Thermo).
8
Quantitative Analysis of DNA Recombination
9
Northern Blot Analysis of SCGB3A2 Expression
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Total RNA (3 μg) isolated from adult lungs of wild type and Scgb3a2-transgenic mice was electrophoresed on 1 % agarose gel containing 0.22 M formaldehyde and transferred onto nitrocellulose membrane (Immobilon-Ny+, Millipore, Billerica, MA). Filters were hybridized with SCGB3A2 probe obtained from Eco RI digestion of the SCGB3A2/pCR2.1 construct. Hybridization was performed in Perfect Hybridization solution (GE Healthcare Life Sciences, Piscataway, NJ) at 68 °C overnight. The membrane was washed twice with 2 x SSC containing 0.1 % SDS at 68 °C for 30 min, followed by exposure to a phosphoimager screen (Storm 840, GE Healthcare Life Sciences, Piscataway, NJ). Data processing was carried out using ImageQuant TL 2005 software (GE Healthcare Life Sciences).
10
Immunoblot Analysis of CREB3, HSV-1, Syndecan-1, and HPSE
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Supernatants of infected HCE cells were vacuum filtered through an Immobilon-Ny+ nylon membrane (Millipore Corporation, Billerica, MA, USA) using the Bio-Dot SF apparatus (Bio-Rad, Hercules, CA, USA). The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies. Detection was accomplished with the ImageQuant LAS 4000 imager (GE Healthcare Life Sciences) as above.
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