SDS-PAGE fractionated gel samples were transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad) according to the manufacturer's protocol. Membranes were then incubated overnight at 4 °C with 20 ml of PBS blocking buffer (4 mM KH2PO4, pH 7.4, 16 mM Na2HPO4, 115 mM NaCl). The blocking buffer was discarded, and the membranes were washed three times with 20 ml of PBS-Tween 20 buffer (PBS buffer with 0.1% v/v Tween 20). Strep-Tactin horseradish peroxidase (HRP) conjugate (IBA Lifesciences GmbH, Germany) diluted 1:100 in enzyme buffer (PBS with 0.2% BSA and 0.1% Tween 20) was added to the membrane and incubated for an hour at room temperature. The membrane was then washed twice using PBS-Tween20 buffer, and twice with PBS. The membrane was incubated for 5 min with 10 ml of peroxide/luminol enhancer solution and imaged using a chemiluminescent imager (GE Healthcare - Imager 600) according to the manufacturer's protocol.
Strep tactin horseradish peroxidase hrp conjugate
Manufactured by IBA Lifesciences
Sourced in Germany
Strep-Tactin horseradish peroxidase (HRP) conjugate is a streptavidin-based detection reagent that can be used to bind and label biotinylated molecules. It consists of streptavidin conjugated to the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Imager 600, by GE Healthcare (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Amersham protran 0.2 m nitrocellulose membranes, by GE Healthcare (1 mentions)
Protran, by Cytiva (1 mentions)
2 protocols using strep tactin horseradish peroxidase hrp conjugate
1
Western Blot Protein Detection
2
Purification of StrepII-tagged PATL2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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).
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