NifH | Nitrogenase iron protein

AS01 021A  |  Clonality: Polyclonal  |  Host: Hen  |  Reactivity: [global antibody]  for bacteria

Benefits of using this antibody

NifH | Nitrogenase iron protein in the group Antibodies Plant/Algal  / Global Antibodies at Agrisera AB (Antibodies for research) (AS01 021A)
NifH | Nitrogenase iron protein


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Product name, number (Agrisera, Sweden)

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Product Information


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

Host Chicken
Clonality Polyclonal
Purity Immunogen affinity purified IgY.
Format Liquid
Quantity 200 ĩg
Storage Store at 4°C; make aliquots to avoid working with a stock. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Tested applications Immunofluorescence (IF), Immunohistochemistry (IHC), Western blot (WB)
Recommended dilution 1 : 500 (IHC), 6 µg/ml (IF), 1 : 2000 (WB)
Expected | apparent MW 27 | 32,5 kDa


Confirmed reactivity Anabaena PCC7120, Clostridium butyricum, Codakia orbicularis, Cylindrospermopsis raciborskii CS-505, Dolichospermum sp., Nostoc sp, Rhodopseudomonas palustris, Trichodesmium sp., nodules of Trifolium repens L., Vibrio natriegens ATCC 14048
Predicted reactivity

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

Species of your interest not listed? Contact us
Not reactive in

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

Application examples

Application examples

Application example


western blot using anti-NifH hen antibody

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).

immunolocalization using anti-NifH antibodies

Immunofluorescence image confirming the NifH protein (bright red dots) close to the cuticle of the ileum and covering or being directly adjacent to the bacterial DNA signals (blue dots: stained by DAPI). The host DNA of the epithelium (e) was also visible. The inset frames show magnifications of red stained dots representing NifH and DAPI signals. Scale bar is10 µm.

Courtesy of Dr. Panagiotis Sapountzis, Center for Social Evolution University of Copenhagen, Danmark

Additional information

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

Related products

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

Product citations

Selected references Li et al. (2022), The effects of Ni availability on H2 production and N2 fixation in a model unicellular diazotroph: The expression of hydrogenase and nitrogenase. Limnol Oceanogr, 67: 1566-1576.
He et al. (2021) Vegetative cells may perform nitrogen fixation function under nitrogen deprivation in Anabaena sp. strain PCC 7120 based on genome-wide differential expression analysis. PLoS One. 2021 Mar 4;16(3):e0248155. doi: 10.1371/journal.pone.0248155. PMID: 33662009; PMCID: PMC7932525. (Immunolocalization)
Liu et al. (2020). A VIT-like transporter facilitates iron transport into nodule symbiosomes for nitrogen fixation in soybean. New Phytol . 2020 Mar 2. doi: 10.1111/nph.16506.
Geisler et al. (2019). Direct Detection of Heterotrophic Diazotrophs Associated with Planktonic Aggregates. Sci Rep. 2019 Jun 26;9(1):9288. doi: 10.1038/s41598-019-45505-4. (immunolocalization)
Murukesan et al. (2019). Acclimation responses of immobilized N2-fixing heterocystous cyanobacteria to long-term H2 photoproduction conditions: carbon allocation, oxidative stress and carotenoid production. J Appl Phycol (2019) 31: 131.
Konig et al. (2016). Nitrogen fixation in a chemoautotrophic lucinid symbiosis. Nat Microbiol. 2016 Oct 24;2:16193. doi: 10.1038/nmicrobiol.2016.193.
Liberti et al. (2015). Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus-growing ant hosts. Mol Ecol. 2015 Apr 24. doi: 10.1111/mec.13216.
Calusinska et al. (2015). Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009. Biotechnology for Biofuels (2015) 8:27.
Moirangthem et al. (2014). A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133. Appl Microbiol Biotechnol. 2014 Jan 3.
Chen et al. (2013). Improving conversion efficiency of solar energy to electricity in cyanobacterial PEMFC by high levels of photo-H2 production. Int J of Hydrogen Energy (2013):1-8.(Anabaena, western blot)
Plominsky et al. (2013). Dinitrogen Fixation Is Restricted to the Terminal Heterocysts in the Invasive Cyanobacterium Cylindrospermopsis raciborskii CS-505. PLOS ONE, Open Access.

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