For core particles, 50 µg each of total cellular proteins were resolved in 1.2% agarose in Tris-buffered EDTA, as described previously [Melegari et al., 2005 (link)], and transferred to nylon membranes (Ambion, Austin, TX) in 10mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM EDTA (TNE buffer). Blots were probed with polyclonal anti-HBc (1:500; Dako Corp., Carpinteria, CA), incubated with peroxidase-conjugated IgG (Sigma Chemical Co.), and detected by chemiluminescence (Perkin Elmer Life Sciences, Waltham, MA). For HBV DNA Southern blot, cells were lysed in 50mM Tris-HCl, pH 8.0, 10mM EDTA, 150mM NaCl, and 1% SDS. Protein-detergent complexes were precipitated in 2.5M KCl, dissolved in Tris-EDTA, and digested by proteinase K (Ambion) for 1 hr at 37 °C. Viral DNA was extracted with phenol-chloroform and precipitated in ethanol followed by electrophoresis of 20 µg total DNA in 1.2% agarose gels. Full-length nonradioactive HBV DNA probes were generated by EcoRI digestion of pCP10 plasmid [Dubois et al., 1980 (link)] using Psoralen-Biotin labeling (Ambion). After denaturation and neutralization, DNA was transferred to nylon membranes (Ambion), crosslinked by ultraviolet light, and hybridized with HBV DNA probe as described previously [Kumar et al., 2012 ].
Nylon membrane
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain
The Nylon membrane is a laboratory equipment product designed for various filtration and separation applications. It is a synthetic material made of nylon polymer, providing a porous structure that allows for the passage of liquids and small particles while retaining larger molecules or contaminants. The Nylon membrane is widely used in the field of biochemistry, molecular biology, and analytical chemistry for tasks such as sample preparation, buffer exchange, and analyte purification. Its core function is to facilitate efficient filtration and separation processes in a controlled and reliable manner.
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Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (20 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (8 mentions)
Lightshift chemiluminescent rna emsa kit, by Thermo Fisher Scientific (5 mentions)
Ecl plus, by GE Healthcare (4 mentions)
Pierce biotin 3 end dna labeling kit, by Thermo Fisher Scientific (3 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (3 mentions)
Biotin 3 end dna labeling kit, by Thermo Fisher Scientific (3 mentions)
Chemiluminescent emsa kit, by Beyotime (3 mentions)
Uv stratalinker 1800, by Agilent Technologies (3 mentions)
Ne per nuclear and cytoplasmic extraction reagent kit, by Thermo Fisher Scientific (3 mentions)
Dna retardation gel, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Unlabeled rna oligonucleotides, by Merck Group (2 mentions)
Human fxr1 gst protein, by Novus Biologicals (2 mentions)
Purified human gst protein, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (2 mentions)
Lightshifttm chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Semi dry transfer apparatus, by Bio-Rad (2 mentions)
Chemiluminescent nucleic acid detection module kit, by Thermo Fisher Scientific (2 mentions)
73 protocols using nylon membrane
1
HBV Core Particle and DNA Detection
2
Quantification of Phosphorylated Signaling Proteins
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Western blotting was performed as described previously [12] (link), [31] (link). Briefly, proteins (25 μg/lane) from the ischemic and non-ischemic hemisphere were size-fractionated on SDS-Page gels, and transferred to nylon membranes (Life Technologies, CA). Membranes were blocked and incubated with primary antibodies for either phosphorylated Akt or ERK (1∶200, SCBT, CA) followed by incubation with HRP-conjugated secondary antibodies (1∶2000, Transduction laboratories, KY). Blots were visualized using the Alpha Innotech imager in conjunction with the ECL detection method. Thereafter, the blots were stripped and reprobed with the pan-antibody for Akt and ERK. In each case, the intensity of ERK and Akt positive bands were quantified using FluorChem software, and phosphorylated protein signal was normalized to the appropriate pan antibody signal.
For detection of heavy (H) and light (L) chain IgG, proteins (25 μg/lane) from the ischemic and non-ischemic hemisphere were size-fractionated on SDS-Page gels, and transferred to nylon membranes (Life Technologies, CA) as before. Blots were incubated with HRP-conjugated anti-rat IgG antibody (1∶1000,Vector Laboratories, CA) and processed as above to visualize the immunoreactive signal. The IgG immunoreactive signal was quantified and intensity of the band of the ischemic hemisphere was normalized to the non-ischemic hemisphere.
For detection of heavy (H) and light (L) chain IgG, proteins (25 μg/lane) from the ischemic and non-ischemic hemisphere were size-fractionated on SDS-Page gels, and transferred to nylon membranes (Life Technologies, CA) as before. Blots were incubated with HRP-conjugated anti-rat IgG antibody (1∶1000,Vector Laboratories, CA) and processed as above to visualize the immunoreactive signal. The IgG immunoreactive signal was quantified and intensity of the band of the ischemic hemisphere was normalized to the non-ischemic hemisphere.
3
Characterization of DNA-Protein Interactions
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EMSA was carried out using the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. NE-PER® Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc.) was used to prepare nuclear extracts from H9c2 and HEK-293 cells, the concentrations of nuclear extract were measured by Bradford protein assay. Double-stranded oligonucleotide fragments (30 bp) containing DSVs were biotinylated and used as probes (Table 1 ). Nuclear extracts (3.0 μg) and probes (0.2 pmol) were incubated for 20 min at room temperature, separated on a native 6% polyacrylamide gel at 100 V for 90 min, and then transferred onto a nylon membrane (Thermo Fisher Scientific, Inc.) at 380 mA for 30 min. UV Stratalinker 1800 (Stratagene, Santa Clara, CA, USA) was used to cross-link oligonucleotides onto the nylon membrane, and the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc.) was used for chemiluminescence detection.
4
Quantifying Transcription Factor Binding
5
EMSA for NF-κB Transcription Factor Binding
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EMSA was conducted using a LightShift chemiluminescent EMSA kit (Thermo Fisher) according to the manufacturer’s instructions using the following biotin end-labeled duplex DNA oligonucleotide: 5′-AAGTGTTTCACTCTGGATCTTATCCTTCAAAGGTCCACTTTTAAAAATTT-3′. For supershift experiments, nuclear extracts were preincubated with one of the following antibodies: anti-NFKB1 (catalog no. 13586; RRID:AB_2665516 ), anti-NFKB2 (catalog no. 3017; RRID:AB_10697356 ), anti-REL (catalog no. 67489; RRID:AB_2799726 ), or anti-RELA (catalog no. 8242; RRID:AB_10859369 ) from Cell Signaling or anti-BCL3 (catalog no. PA5-28783; RRID:AB_2546259 ) from Thermo Fisher. Binding reactions were resolved on a 3% to 12% NativePAGE Bis-Tris gel (Thermo Fisher) and transferred to nylon membranes (Thermo Fisher).
6
PARP9 Binding to Reovirus dsRNA
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Aliquots of cell-free PARP9 recombinant protein (0.5, 1, 5 μg) were mixed with 1 pmol of biotinylated reovirus dsRNA Reo1198 (Reo1198) or unlabeled viral dsRNA Reo1198 (1, 5, 20 μg) for competitor assay, and incubated at room temperature for 1 h. Controls included, protein only and biotinylated-Reo1198 only reactions. After incubation for 1 h, proteins and biotinylated Reo1198 dsRNA were crosslinked with 1% formaldehyde for 30 min on ice. Crosslinking was quenched by adding glycine to a final concentration of 125 mM. Reactions were subjected to gel electrophoresis (Novex TBE-Urea 10% Gels, Invitrogen, Carlsbad, CA, USA) and transferred to nylon membranes (ThermoFisher Scientific). Biotin-labeled dsRNA was detected using the LightShift Chemiluminescent RNA EMSA Kit (ThermoFisher Scientific)63 (link),66 (link).
7
Nuclear Protein-DNA Binding Assay
8
Electrophoretic Mobility Shift Assay for ZFP24
9
Western Blot Analysis of Protein Expression
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After collection, HK-2 cells and renal tissues were lysed using RIPA buffer (89,900, Thermo Fisher Scientific, MA, USA) containing 0.1 M phenylmethylsulphonyl fluoride (PMSF, 36,978, Thermo Fisher Scientific, MA, USA). The supernatant was collected after pre-clear by centrifugation (12,000 g × 15 min) at 4℃. Protein concentration was determined using a BCA protein assay kit (23, 227, Thermo Fisher Scientific, MA, USA) . 20 µg of total proteins for each sample were subjected to 12% SDS-PAGE and transferred to Nylon membranes (LC2003, Thermo Fisher Scientific, MA, USA). After blocking with 5% nonfat milk in 1 × Tris-buffered saline with 0.1% Tween® 20 Detergent (TBST), the membranes were incubated with the corresponding primary antibodies (shown in Table 1). After three washes by 1×TBST, the membranes were incubated with the corresponding horseradish peroxidase-labeled secondary antibody (shown in Table 1). The membrane was developed using an ECL substrate kit (ab65623, Abcam, MA, USA). The protein bands were analyzed by Image Lab Software 6.1 (Bio-Rad, CA, USA).
10
Electrophoretic Mobility Shift Assay for ZFP24
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EMSA was performed using the LightShift Chemiluminescent EMSA kit (Pierce). Nuclear extract derived from HEK cells transfected with a ZFP24 encoding plasmid was incubated with biotinylated probe and run on 5% TBE PAGE gel. The gels were then transferred to nylon membranes (Thermo Fisher Scientific). Biotin-labeled probes were detected by primary streptavidin antibody attached to horseradish peroxidase. Biotinylated probes used were as follows: TCAT probe: 5′-TCATTCATTCATTCATTCATTCATCAT-3′; scramble probe: 5′-AGAGTGGTCAATACCCCCTCTG-3′; Mbp probe: 5′-GTT TTG TTA ATG CAT TTA ATT CCT CAC AGT ATA GCT GTT GTC TTC TCT CCT CAT CTG CGT TCG TTT CTG ACA GCT ACA GAA TTT AT-3′; Sox10: 5′-GGT AGT TGC TTC ATT CAT TCA TTC ACT CAT TCA TTC ATT CAT TCA TTC ATT CAT TCA TTC ATC TTG GAG TTT CCT TTA GTA CA-3′. For reactions that contained the unlabeled “cold” probe, the same unlabeled probe was added to the reaction in 200-fold excess.
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