ADH | Alcohol dehydrogenase (hypoxia marker)

KLH-conjugated peptide derived from available ADH sequences including Arabidopsis thaliana P06525, At1g77120

42 | 42 kDa (Arabidopsis thaliana)

Application example

20 µg of total protein from Arabidopsis thaliana seedlings (0-4-8 hours of anoxic treatment with aerobic control) of WT Col-0 and adh mutant extracted with an SDS Extraction Buffer (60mM Tris-HCl pH 8.0, 2% SDS, 1,5% Sucrose) were separated on XT CRITERION 10%Bis-Tris (BioRad) SDS-PAGE and blotted 1h to PVDF. Blot was blocked immediately in milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the anti-ADH antibodies at a diluition of 1: 3000 in milk in TBS-T for 3h at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG HRP conjugated from Agrisera, AS09 602) diluited 1:20 000 in milk in TBS-T for 50 min at RT and then washed as above and developed for 2 min with chemiluminescent detection reagent. Images of the blot were obtained using BioSpectrum AC Imaging System (UVP). Exposure time was 10 min The arrow indicates ADH (42kDa, as expected) . There is a cross reacting band in Arabidopsis thaliana between 50-70 kDa. The large band in the right corner of the membrane is likely a staining artefact.

20 µg of total protein from Orysa sativa coleoptiles (3-4-5-6 days of germination under aerobic and anoxic conditions) extracted with an SDS Extraction Buffer (60mM Tris-HCl pH 8.0, 2% SDS, 1,5% Sucrose) were separated on XT CRITERION 10% Bis-Tris (BioRad) SDS-PAGE and blotted 1h to PVDF. The blot was blocked immediately in milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the anti-ADH antibodies at a diluition of 1: 3000 in milk in TBS-T for over night with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG HRP conjugated from Agrisera, AS09 602) diluted 1:20 000 in milk in TBS-T for 50 min at RT and then washed as above and developed for 2 min with chemiluminescent detection reagent. Images of the blot were obtained using BioSpectrum AC Imaging System (UVP). Exposure time was 10 min. The band corresponds to ADH (41 kDa).

Courtesy Dr. Eleonora Paparelli,Scuola Superiore Sant'Anna, Italy

AS10 691 | Anti-PDC | pyruvate decarboxylase, rabbit antibodies

Plant protein extraction buffer

Secondary antibodies

Alcohol dehydrogenase (ADH) is an enzyme playing a crucial role in the fermentative metabolism in plants subjected to low oxygen stress. It is known to be synthesized preferentially under low oxygen conditions.