AtpB | positive control/quantitation standard
AS03 030S | recombinant protein standard for quantitation and positive control
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Standard curve: 3 loads are recommended (0.5, 2 and 4μl).
Positive control: load per well: a 2μl load is optimal for most chemiluminescent detection systems.
This standard is stabilized and ready and does not require heating before loading on the gel.
|Expected | apparent MW||
in most gel systems AtpB migrates around 50-54 kDa
|Not reactive in|
Concentration: after adding 225 µl of dest. water final concentration of the standard is 0.27 pmol/µl.
Fraser et al. (2013). Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. PLOS ONE.
AtpB protein standard (AS03 030S) 0.03, 0.1, 0.26 pmol (1-3) and total proteinfrom Trichodesmium IMS 101 extracted with PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horseradish peroxidase conjugated, from Abcam) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturer's instructions. Images of the blots were obtained using a CCD imager nd Quantity One software Exposure time was 10 seconds.
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