NifH | nitrogenase iron protein

371 €

AS01 021A  |  clonality: polyclonal  |  host: hen  |  reactivity: [global antibody]  for bacteria


22 st
Item No:
AS01 021A

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product information

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


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 Hen
Clonality Polyclonal
Purity Affinity purified IgY
Format Liquid in PBS pH 8.0, 0.02% sodium azide
Quantity 200 ĩg

store at 4°C; make aliquots to avoid working with a stock. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from liquid material adhering to the cap or sides of the tubes.

Tested applications western blot (WB), immunofluorescence (IF)
Related products

AS01 021S | NifH protein standard for a quantitative western blot
Recommended secondary antibody for chemiluminescent detection, AS10 1489
Recommended secondary antibody for other detection systems

Algal protein extraction buffer

Secondary antibodies

Additional information

antibody concentration is 1.36  µg/µl


application information
Recommended dilution

1: 2 000 (WB), 6 µg/ml (IF)

Expected | apparent MW

27 | 32.5 kDa

Confirmed reactivity

Anabaena PCC7120, Clostridium butyricum, Cylindrospermopsis raciborskii CS-505, Nostoc sp, Rhodopseudomonas palustris, Trichodesmium sp., nodules of Trifolium repens L.

Predicted reactivity

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

Not reactive in

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

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

Selected references 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.

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% 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) 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).

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

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