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 90 µl of dest. water final concentration of the standard is 0.27 pmol/µl.
|Selected references||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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