PsaE | PSI-E subunit of photosystem I (cyanobacterial)
AS19 4355 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Synechocystis sp. PCC 6803, Thermosynechococcus elongatus
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Soluble (8 μg protein) and membrane proteins (corresponding to 1 μg of chlorophyll a) from Synechocystis sp. PCC 6803
1 – WT (soluble)
2 - DPsaE – PsaE deletion (soluble)
3 - EY10 – PsaE-HoxY fusion (10 aa linker) (soluble)
4 - EY16 – PsaE-HoxY fusion (16 aa linker) (soluble)
5 - EU – PsaE-HoxU fusion (soluble)
6 – WT (membrane)
7 - DPsaE – PsaE deletion (membrane)
were extracted with and resuspended in ACA buffer (750 mM e- amino caproic acid; 50 mM BisTris/HCl, pH 7.0; 0.5 mM EDTA). Samples were denatured with 2x sample buffer (125 mM Tris, pH=6,8; 200 mM DTT; 4% (w/v) SDS; 20% (w/v) Glycerin; 0,02% (w/v) bromophenol blue) at room temperature (RT) for 1h. The proteins were separated on 17.5 % SDS PAGE (Bis-Tris) gels and blotted for 60 min onto a nitrocellulose membrane using a wet transfer system (BioRad). The membrane was blocked with 5% milk powder in PBS-T for 1 h at RT with agitation. The blot was then incubated overnight with the primary antibody at a dilution of 1:5.000 in PBS-T at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 20 min in PBS-T with agitation. The blot was incubated using a matching secondary antibody (anti-rabbit IgG horseradish peroxidase conjugated) diluted to 1:10.000 in PBS-T for 1 h at RT with agitation. The blot was washed three times for 10 min with PBS-T and two times for 10 minutes with PBS. Subsequently the membrane was incubated with chemiluminenscent detection reagent. The differently exposed films were developed using a NDT DÜRR developer.
Courtesy of Dr.Marko Boehm,Botanisches Institut der Christian-Albrechts-Universität zu Kiel, Germany
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