FtsH1-11 | ATP-dependent zinc metalloprotease FtsH1-11
AS11 1789 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. acetabulum, A. thaliana, B. hypnoides (ulvophytes), G. theta, H. vulgare, Synechocystis sp., T. pseudonana, Z. mays
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1 : 500 - 1: 2000 (WB)
|Expected | apparent MW||
A. thaliana: FtsH1:76.7, FtsH2: 74, FtsH5: 75.2, FtsH6: 74.5, FtsH7: 87.8, FtsH8: 73, FtsH9:87.8 kDa (chloroplastic); FtsH3: 89.3, FtsH4: 77.2, FtsH10: 89.5 kDa, FtsH11:88.7 kDa (mitochondrial)
|Confirmed reactivity||Acetabularia acetabulum, Arabidopsis thaliana, Bryopsis hypnoides (ulvophytes), Guillardia theta, Hordeum vulgare, Synechocystis sp., Thalassiosira pseudonana, Thalassiosira punctigera, Zea mays|
di and monocots, Chlamydomonas reinhardtii
|Not reactive in||
No confirmed exceptions from predicted reactivity known in the moment
For detection on a diatom samples, load of 5-10 µg/well is required as well as usage of enchanced chemiluminescence.
The antibody also detects the chloroplast-encoded FtsH from Thalassiosira pseudonana (diatom) along with 3 related isoforms encode in the nuclear genome of Thalassiosira pseudonana.
For detection in BlueNative, recommened dilution is 1: 500.
|Selected references||Li et al. (2016). A Hard Day's Night: Diatoms Continue Recycling Photosystem II in the Dark. Front. Mar. Sci., 08 November 2016
Tietz et al. (2015). Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes. J Biol Chem. 2015 Apr 20. pii: jbc.M114.619841.
Campbell et al. (2013). Photosystem II protein clearance and FtsH function in the diatom Thalassiosira pseudonana. Photosynth. Res. March 16.
8 µg of total protein from Arabidopsis thaliana wild type (1) and FtsH 1 and 3 deletion mutant (2) extracted with 0.2M Tris-HCl pH6.8, 2%SDS, 10% mercaptoehanol, 5M Urea were separated on 12 % SDS-PAGE and blotted 2h to NC. Blots were blocked with non fat milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 over night at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 5 min and 2 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody anti-rabbit IgG horse radish peroxidase conjugated, from Sigma) diluted to 1:10 000 in TBS-T+5% non fat milk for 2h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 3 minutes.Courtesy of Dr. Leah Nave, The Hebrew University, Israel
|The dillution series of Hordeum vulgare (4; 2; 1 µg Chl) and Arabidopsis thaliana (3,6; 1,8; 0,9 µg Chl) thylakois were separated on 12% Criterion XT Bis-Tris SDS-PAGE (BioRad) gels and blotted for 25min 100V to PVDF membrane. Blot was blocked with 5% fat free skimmed milk in PBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 overnight with agitation in 4˚C. The antibody solution was decanted and the blot was washed 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (swine anti-rabbit IgG horse radish peroxidase conjugated, from Dako) diluted to 1: 5000 in 1% fat free skimmed milk in PBS-T for 1h at RT with agitation. The blot was washed 5 min in PBS-T and 1 min in PBS developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 90 seconds.
The band below FtsH2 is suspected to be FtsH5 as according to Sinvany-Villalobo et al. (2004): The most abundant FtsH transcript is that of FtsH2 (Fig. 3B), demonstrating a level similar to ClpC1. FtsH1 and 5 are also relatively abundant, each accumulating to levels of about 50% to 60% of FtsH2 transcript. Other FtsH transcripts are much less abundant, with FtsH6 not accumulating at all under optimal growth conditions.
Courtesy of Dr. Marta Powikrowska, University of Copenhagen, Danmark
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