BL21 (DE3) pLysS-containing pET-52b(+) vectors with recombinant PATL2 or PATL2 deletion mutants were grown in 50 ml LB (100 µg/ml ampicillin, 34 µg/ml chloramphenicol, OD600 of 0.08). The temperature was lowered from 37 to 30 °C when the culture reached an OD600 of 0.3–0.4. Protein expression was induced 30 min after the temperature shift with 1 mM isopropyl-ϐ-d -thiogalactopyranosid (IPTG) and cells were harvested 3 h later. StrepII-tagged protein of interest was purified using the Strep-tag®/Strep-Tactin® system (IBA Lifesciences) according to the manufacturer’s protocol for Strep-Tactin® Macroprep® or via Strep-Tactin®XT Superflow® cartridge using ÄKTA prime plus (GE Healthcare Life Sciences). Protein expression and purification were assessed following standard SDS–polyacrylamide gel electrophoresis, either followed by Coomassie Brilliant Blue staining or by blotting on Amersham™ Protran™ 0.2 µm nitrocellulose membranes (GE Healthcare Life Sciences) and affinity detection via enhanced chemiluminescence (GE Healthcare Life Sciences) of StrepII-tagged protein using Strep-Tactin®-horseradish peroxidase (HRP) conjugate (Iba Lifesciences).
Amersham protran 0.2 m nitrocellulose membranes
Manufactured by GE Healthcare
Sourced in United States
Amersham™ Protran™ 0.2 µm nitrocellulose membranes are laboratory equipment used for protein transfer and immobilization in Western blot analysis. These membranes have a pore size of 0.2 microns and are made of nitrocellulose material.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Strep tactin horseradish peroxidase hrp conjugate, by IBA Lifesciences (1 mentions)
Cell lysis buffer 10, by Cell Signaling Technology (1 mentions)
Tris glycine sds running buffer, by Bio-Rad (1 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
2 protocols using amersham protran 0.2 m nitrocellulose membranes
1
Purification of StrepII-tagged PATL2 Proteins
2
Synthesis and Purification of Indolyl-Benzimidazoles
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SY-LB-35 and SY-LB-57 are indolyl-benzimidazoles that were synthesized and purified by HPLC by Dr. Leonard Barasa in the laboratory of Dr. Sabesan Yoganathan at St. John's University (Queens, NY, USA). Details of SY-LB synthesis, purification and characterization were previously reported [30, 31] . Cell Lysis Buffer (10×) was obtained from Cell Signaling Technologies (Danvers, MA, USA). TGX Fast Cast Acrylamide gel kit (12%), 4× Laemmli Sample Buffer, 10× Tris/Glycine/SDS Running Buffer and Precision Plus Protein Standards were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Amersham™ Protran™ 0.2 µm Nitrocellulose membranes were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Bovine Serum Albumin (30% solution) was from Alfa Aesar (Ward Hill, MA, USA). SuperSignal™ West Femto HRP Substrate solutions for chemiluminescent imaging was from Thermo Scientific (Rockford, IL, USA).
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