Plant/Algal cell antibodies / Photosynthesis / Protein standards-quantitation

product information

ATP synthase is the universal enzyme that stnthesizes ATP from ADP and phosphate using the energy stored in a transmembrane ion gradient.

This product is a recombinant protein standard, source: Synechocystis strain PCC 6803. 

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store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Western blot (WB)

collection of other protein standards

AS03 030 | anti-ATP synthase subunit beta global antibody (hen)

AS05 085 | anti-ATP synthase subunit beta global antibody (rabbit)

collection of other global antibodies

global antibodies are raised against highly conserved amino acid sequences in the AtpB protein. The AtpB protein standard can therefore be used in combination with global anti-AtpB antibodies to quantitate AtpB from a wide range of species.

Quantitative western blot:  detailed method description.

application information

standard curve: 3 loads are recommended (0.5, 2 and 4μl).
For most applications a sample load of 0.2μg of chlorophyll will give a AtpB signal in this range.

positive control: load per well: a 2μl load is optimal for most chemiluminescent detection systems.

This standard is stabilized and does not require heating before loading on the gel.

in most gel systems AtpB migrates around 50-54 kDa

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no confirmed exceptions from predicted reactivity known in the moment

Concentration: after adding 225  µl of dest. water final concentration of the standard is 0.27 pmol/µl

Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50mM DTT.

Brown et al. (2007) Resource dynamics during infection of Micromonas pulsilla by virus MpKSp1. Environmental Microbiology 11: 2720-2727.

application example

AtpB protein standard (AS03 030S) 0.03, 0.1, 0.26 pmol (1-3) and total protein from 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.
Note: Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.