ADH/ALDH | Alcohol/acetaldehyde dehydrogenase (bacterial/algal)

KLH-conjugated peptide derived from available algal and bacterial ADH sequences including Chlamydomonas reinhardtii

100 | 100 kDa (C.reinhardtii), 96 kDa (E.coli)

Application example

30 μg of a total cell extract from Chlamydomonas reinhardtii and E.coli strains DC272 and DC271 were loaded on Criterion™,Tris-HCl 10% polyacrylamide gels (Biorad) and molecular weight compared to those of the PageRuler™ Plus Prestained Protein Ladder (Fermentas). After SDS-PAGE, gels were transferred to PVDF membranes (Biorad) by the Trans-Blot SD semidry Transfer Cell method (Biorad) for 1 hour at 10V. Blocking of the PVDF membrane has been done for 3 hours in TBST milk 5% and has been followed by overnight incubation at 4°C with the primary anti-ADH/ALDH antibodies 1:1000 in TBST milk 1%. After three intensive washes, the membrane was incubated for one hour at room temperature with the secondary HRP-conjugated goat anti-rabbit (Agrisera AS09 602, in 1:50 000 dilution in TBST milk 1%). After three washes with TBST (10 minutes each), detection was achieved using chemiluminescent detection reagent. Exposure time was 2 minutes for Chlamydomonas reinhardtii sample and 10 seconds for E. coli samples.

E. coli strains DC272 and DC271 were provided by Professor David P. Clark, Souther Illinois University. The DC272 mutant strain is misregulated in AdhE expression so that the bacteria expresses the ADHE protein constitutively.

Courtesy Dr. Leonardo Magneschi, Scuola Superiore Sant'Anna, Italy
 

Alcohol dehydrogenase (E.C.:1.1.1.1) is an important enzyme for plants and microbes. In microalgae and bacteria the conversion of Acetyl-CoA to ethanol under conditions of oxygen deprivation is catalyzed by the dual function enzyme alcohol/acetaldehyde dehydrogenase (ADH/ALDH; E.C.:1.1.1.1 /1.2.1.10). This reaction results in NAD+ recycling and allows glycolysis to proceed.