Protran ba 85 membrane

For SDS/PAGE, approximately 100 µg denatured protein of the bacterial lysate supernatants were analyzed on a 7.5% polyacrylamide gel. The separated protein bands were then transferred onto a Protran BA 85 Membrane (Carl Roth GmbH, Karlsruhe, Germany) and the blots were probed with an anti-his-tag-HRP conjugated antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Immunoreactive bands were visualized using the SERVALight Polaris CL HRP WB Substrate Kit (Serva Electrophoresis GmbH, Heidelberg, Germany). Chemiluminescence was detected on a FUJIFILM Luminescent Image Analyzer LAS-1000plus & Intelligent Dark Box II. For testing the pH-dependence of the product mixture of wildtype human and mouse ALOX15B orthologs, a 1:1 mixtures of 0.05 M sodium phosphate buffer and 0.05 M sodium borate buffer were used and the different pH values were adjusted at room temperature by the addition of 5 M NaOH or HCl, respectively. The protein concentrations in the bacterial lysates were quantified using Bradford Reagent for quantitative protein determination (AppliChem, VWR International GmbH, Darmstadt, Germany) according to the instructions of the vendor. For statistical calculations and figure design we used the GraphPad prism program (version 8.00, GraphPad Software, La Jolla, CA, USA).

For SDS-PAGE analysis, 100 µg denatured protein of the bacterial lysate supernatants were run on a 7.5% polyacrylamide gel. The proteins were then transferred onto a Protran BA 85 Membrane (Carl Roth GmbH, Karlsruhe, Germany) and the blots were probed with an anti-His-HRP antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Immunoreactive bands were visualized using the SERVALight Polaris CL HRP WB Substrate Kit (Serva Electrophoresis GmbH, Heidelberg, Germany). Chemiluminescence was detected on a FUJIFILM Luminescent Image Analyzer LAS-1000plus & Intelligent Dark Box II. The protein concentrations in the bacterial lysates were quantified using Bradford Reagent for quantitative protein determination (AppliChem, VWR International GmbH, Darmstadt, Germany) according to the instructions of the vendor. Amino acid sequence alignments were performed using the EMBOSS Needle tool (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 25 April 2019).