AtpB | Positive control/quantitation standard

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 ready and does not require heating before loading on the gel.
Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

in most gel systems AtpB migrates around 50-54 kDa

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 Agrisera Protein Extraction Buffer (AS08 300) were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent 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) diluted to 1:50 000 in 2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescence 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.

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

Quantitative western blot:  detailed method description, video tutorial

Concentration: after adding 90 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.

This standard is ready-to-load and does not require any additions or heating. It needs to be fully thawed and thoroughly mixed prior to using. Avoid vigorous vortexing, as buffers contain detergent. Following mixing, briefly pulse in a microcentrifuge to collect material from cap.
This standard is stabilized and ready and does not require heating before loading on the gel.
Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

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.