NifH | positive control/quantitation standard
AS01 021S | recombinant protein standard for quantitation of Nifh protein
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Standard curve: 3 loads are recommended (0.5, 2 and 4 μl).
Positive control: a 2 μl load per well 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||
34 kDa (larger than a native protein due to the addition of His-tag)
|Not reactive in||
no confirmed exceptions from predicted reactivity known in the moment
Concentration: after adding 225 µl of milliQ water final concentration of this standard is 0.15 pmoles/ul and this reagent is ready to use and load on a gel.
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.
Levitan et a. (2010). Regulation of nitrogen metabolism in the marine diazotroph Trichodesmium IMS101 under varying temperatures and atmospheric CO concentrations. Environ. Microbiol (Epub ahead of print)
Total Trichodesmium sp. protein extract(lanes 6-11, 80 pmol chlorophyll loaded) extracted with PEB (AS08 300), and NifH protein standard (lanes 1-5, 0.05, 0.1, 0.3 0.75 and 1.5 pmol standard loaded) 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 1:40 000 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 horse radish 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
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.
Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.
Quantitative western blot: detailed method description.
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