Samples were mixed with an equal volume of tricine sample buffer (BioRad, Hercules CA) and then electrophoresed on 12.5% sodium dodecyl sulfate-tris-tricine polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes via electroblotting as previously described [27 (link)]. Membranes were stained with reversible stain Fast Green FCF 0.1% (Fisher Scientific, Waltham, MA) in 25% methanol-10% acetic acid for 1 min, destained with 25% methanol, and then transferred to distilled water to assess equal loading. Membranes were then washed in TBST and blocked for 1 h at room temperature with 5% nonfat dry milk in TBST. Blots were then incubated with PHF1 (1:500), 6E10/4G8 (1:2000), and actin (1:5000; Sigma) overnight at 4 °C. The next day, bound antibodies were detected after 1 h incubation with goat anti-mouse or anti-rabbit IgG HRP (1:3000; GE Healthcare, UK) and the chemiluminescent detection system (Pierce, Rockford, IL) on autoradiography films (MIDSCI, St. Louis, MO).
Autoradiography film
Manufactured by MidSci
Sourced in United States
Autoradiography film is a type of photographic film used to detect and visualize radioactive signals in biological samples. It is designed to capture and record the radioactive emissions from labeled molecules, such as DNA, RNA, or proteins, allowing researchers to analyze the distribution and abundance of these molecules within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Tricine sample buffer, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Silica gel 60 f254 plate, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Phototope star detection kit, by New England Biolabs (1 mentions)
Hybond, by Cytiva (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Pierce ecl detection system, by Thermo Fisher Scientific (1 mentions)
Mini protean electrophoresis system, by Bio-Rad (1 mentions)
5 protocols using autoradiography film
1
SDS-PAGE and Western Blotting Analysis
2
GPI Anchor Autoradiography Protocol
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GPI anchor autoradiography was performed as previously described (17 (link), 57 (link), 59 (link)). Briefly, 33- to 39-h-old parasites that had been grown in ±aTc conditions were washed and resuspended in glucose-free media (RPMI R1383 + 20 mM D-fructose, 25 mM Hepes, 21 mM NaHCO3, 0.37 mM hypoxanthine, 11 mM glutathione, 5 g/l Albumax I, and 10 μg/ml gentamicin). One hundred fifty microcurie of 40 Ci/mmol [3H]GlcN (American Radiochemicals) was added to each 10 ml culture and incubated for 3 h. Parasites were saponin lysed, and glycolipids were extracted with chloroform:methanol:water (10:10:3), nitrogen evaporation, and n-butanol partitioning (60 ). The resultant glycolipids were run on TLC Silica gel 60 F254 plates (Millipore Sigma) using chloroform:methanol:water (10:10:3) as a solvent. The TLC plates were exposed to autoradiography films (MidSci), which were developed after 1 week of exposure. Films were imaged on a BIO-RAD ChemiDoc MP imaging system, and the signal was quantified by the ImageLab software from Bio-Rad.
3
Immunoblotting and Co-immunoprecipitation Protocols
4
DNA Shift Assays of LdtR Mutants
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DNA shift assays of LdtR, as well as its mutated versions, was carried out as described previously [9 (link)]. Fragments of the selected promoters were generated by PCR using biotinylated primers (Table 2 ). The reaction mix for EMSA contained 1 ng of 5’biotin-labelled DNA probe, 50 nM Tris-HCl pH 7.2, 150 mM KCl, 10 mM MgCl2, 0.01% Triton X100, 12.5 ng/μl both Poly(dI-dC) and Poly(dA-dT) nonspecific competitor DNAs, and purified LdtR (0–1000 nM) as indicated. The mix was incubated for 20 min at 37°C and the electrophoresis was conducted at 4°C on 6% acrylamide/bisacrylamide non-denaturing gels in 0.5X Tris-borate EDTA buffer (TBE) pH 8.3. The DNA was transferred to a Hybond-N+ membrane (GE Healthcare, Pittsburgh, PA, USA) by electroblotting at 250 mA for 45 min in a Semi Dry Electroblotting System (Fisher Scientific). The detection of the DNA bands was carried out with the Phototope-Star Detection Kit (New England Biolabs, Ipswich, MA, USA). The membranes were exposed to Autoradiography Film (Mid-Sci, St. Louis, MI, USA).
5
Signaling Assays and Co-Immunoprecipitation Protocol
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For signaling assays, NCI-H1299 and NCI-H1792 cells were seeded at confluence and serum starved overnight. Cells were lysed in RIPA buffer (Sigma) and the lysates Western blotted on a 4-15% Tris-glycine gel and transferred to a 0.2μm nitrocellulose using a Mini-PROTEAN electrophoresis system (Bio-Rad Laboratories, Hercules, CA). For co-immunoprecipitation, HEK 293T cells were transfected as above and lysed in modified RIPA buffer (150mM NaCl, 50mM Tris, pH 7.5, 1% NP-40). Precleared lysates were immunoprecipitated with GFP-Trap agarose beads (Allele Biotech, San Diego CA) or primary antibody as appropriate and washed with lysis buffer. Western blots were developed using a Pierce ECL detection system (Thermo Scientific, Rockford IL) and autoradiography film (MidSci, St. Louis, MO).
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