FtsH10 | ATP-dependent zinc metalloprotease FtsH10 (mitochondrial)

KLH-conjugated peptide located near C-terminus chosen from sequence of Arabidopsis thaliana FtsH10 Q8VZI8, At1g07510

84 kDa

Arabidopsis thaliana

Application example

Mitochondrial preapration from Arabidopsis thaliana mitochondria was separated on 10% acrilamide gel and electrophoresis prepared according to Schägger and von Jagov (Anl. Biochem., 1987, 166:368-379). After running the gel, proteins were transferred to nitrocellulose membrane using wet transfer (0.22% CAPS, pH 11). Transfer was checked by Ponceau S staining. Blot was destained by several quick washings in distilled water and 1 washing in 1X TBS (10 mM T pH 7.5, 150 mM NaCl) (10-15 min.).Blot was blocked by 1.5 hour in 5% milk in TBST (1X TBS, 0,1 20) After blocking blot was washed quickly twice in TBST and incubated 2 hours with primary antibody (dilution 1: 1000 TBST (dilution 1:1000). Washing: two quick washings in TBST and 3 x 10 min. washings in TBST. Then blot was incubated 45-60 min. with a secondary anti-rabbit antibodies conjugated to peroxidase (dilution 1:10000) in TBST. Washing: as above. After washing blot was incubated 1-2 min. in ECL solution and exposed to Kodak autoradiography film. Exposure time was 15-60 seconds.

Mitochondria were isolated as described by Urantowka et al. (Plant Mol Biol, 2005, 59:239-52). Mitochondrial pellets were suspended in 1X Laemmli buffer (5% beta-mercaptoetanol, 3.7% glycerol, 1.1% SDS, 23 mM Tris-HCl pH 6.8, 0.01% bromophenol blue), heated (95 ºC, 5 min.) and centrifuged (13000 rpm, 1 min.).

Courtesy Dr. J. Piechota, University of Wroclaw, Poland

One of the several classes of mitochondrial proteases is membrane bound, ATP dependent FtsH protease. Their function is very important for the control of protein quality and quantity by degradation of unassembled subunits. FtsH10 is localized in mitochondria. Alternative names: cell division protease ftsH homolog 10, mitochondrial, AtFtsH10