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.

tested applications

western blot (WB)

related products

collection of other protein standards

AS01 021A | anti-NifH hen global antibodies

collection of other global antibodies

collection of antibodies to proteins involved in nitrogen metabolism

additional information

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

Quantitative western blot: detailed method description, video tutorial

application information
recommended dilution		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 NifH signal in this range. positive control: a 2μl load per well is optimal for most chemiluminescent detection systems. This standard is stabilized and does not require heating before loading on the gel.
expected \| apparent MW		34 kDa (larger than native protein due to the addition of His-tag)
confirmed reactivity
predicted reactivity
not reactive in		no confirmed exceptions from predicted reactivity known in the moment
additional information		Concentration: after adding 225 µl of milliQ water final concentration of the standard is 0.15 pmoles/ul 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.
selected references		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)

application example

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
according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).

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.

western blot using anti-NifH hen antibody

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.

||| For applications or usage on species others than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions on this product, please use the LiveChat option in the left menue bar or contact us at support@agrisera.com

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