NifH | Nitrogenase iron protein

KLH-conjugated synthetic peptide derived from known bacterial NifH subunits of bacterial nitrogenase enzymes of the FeMoCo type including Synechoccocus sp. Q2JP78 , Trichodesmium theibautii, Anabaena sp. P33178 and Nostoc sp. Q51296

Azotobacter vinelandii (Gram-), Bradyrhizobium japonicum, Cyanobacteria, Cyanothece ATCC51142, Desulfotomaculum reducens (strain MI-1),Clostridium cellobioparum,  Enterobacter , genera,  euryachaeotes, Klebsiella pneumonia, Magnetococcus sp., Methanobacterium thermoautotrophicum, Methanococcus maripaludis, Methylobacterium sp., Mesoorhizobium loti, Rhodopseudomonas palustris TIE-1 strain, alpha,gamma,beta proteobacteria, enterobacteria, low GC gram+, high GC gram +, able to fix atmoshperic nitrogen, Rhizobium meliloti

Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 as NifH protein is not present in those cyanobacterial species, Frankia sp.

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% 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 1:40 000 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) 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 chemiluminescent 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).

An enzyme involved in chlorophyll synthesis, present in all cyanobacteria (fixing and non-nitrogen fixing)  is a member of the NifH family/superfamily. Agrisera anti-NifH antibody will not show a strong reactivity to this target.

In photobionts like Anabaena sp., low nitrate growth is required to turn on the NifH expression to high enough levels to detect NifH protein.

Immunofluorescence protocol
Insect dissected tissues (digestive tract, fat body, carrying NifH positive bacteria) of large workers were fixed in cold methanol (20 min, -20°C) and then permeabilized in cold acetone (5 min, -20°C). Samples were subsequently rinsed three times with PBS with 0.1 % Triton-X 100 at RT (PBST) and incubated for 5 minutes in PBST. This was followed by incubation of tissues for 1 hr with 6 ug/ml affinity purified anti-NifH antibody (Agrisera, AS01 021A) diluted in PBS-TBSA (PBS, 0.1 % v/v Triton-X-100, 1 mg/ml BSA) and 3 washings with PBST. Samples were then incubated in the dark with a goat anti-chicken IgY conjugated to Dylight 488 (Pierce, SA5-10070) for 45 min and were washed twice (PBS, 0.1%v/v Triton-X-100). Finally, the tissues were mounted in Vectashield medium containing DAPI (Vector Laboratories, H-1500) and viewed under a SP5 Leica confocal microscope with 10X and 63X objectives.
Courtesy of Drs. Panagiotis Sapountzis and Mariya Zhukova, University of Copenhagen, Danmark

Nitrogenase is involved in biological fixation of atmospheric nitrogen to ammonia. Alternative protein names: nitrogenase component II, nitrogenase Fe protein, nitrogenase reductase, FeMoCo-nitrogenase.