Dear researcher, I know that nitrogenase is responsible to fix atmospheric nitrogen. Which technique are used to detect the nitrogenase enzyme? Please tell me answer so I will gratitude for everybody.
Dear Noorain,
The following publications describe the detection methods for nitrogenase from bacteria:
1- http://chemwiki.ucdavis.edu/Wikitexts/University_of_California_Davis/UCD_Chem_124A%3A_Berben/Nitrogenase/Nitrogenase_2
EPR spectroscopy is the technique often used to study the nitrogenase and its cluster. EPR experiments show that MoFe cofactor posses a 3/2 spin state.12 Information such as this spin state give a valuable spectroscopic fingerprint that can help detect what will happen to nitrogenase under different circumstances, i.e. what happens to the active site when it is purified or isolated.
2-JOURNAL OF BACTERIOLOGY, Jan. 1991, p. 365-371
Detection of Alternative Nitrogenases in Aerobic Gram-Negative
Nitrogen-Fixing Bacteria
ELAZAR FALLIK,1 YIU-KWOK CHAN,2 AND ROBERT L. ROBSON'*
Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A niJDK gene probe was used as a
control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 238331; ,nd also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than A ,o-Fe N2ase structural
genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anJDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cop
3-https://books.google.ps/books?id=n-XVfskUuI0C&pg=PA93&lpg=PA93&dq=technique++used+to+detect+nitrogenase+enzyme&source=bl&ots=asNFhBEENL&sig=_iio42SVHF9nNAQsFcyyotRzmwI&hl=en&sa=X&ved=0ahUKEwjap8SmksbKAhWI7RQKHfMVCwgQ6AEIPzAG#v=onepage&q=technique%20%20used%20to%20detect%20nitrogenase%20enzyme&f=false
4-https://www.google.ps/search?biw=1366&bih=667&q=related:www.ru.nl/publish/pages/549461/staalem2001.pdf+technique++used+to+detect+nitrogenase+enzyme&tbo=1&sa=X&ved=0ahUKEwjap8SmksbKAhWI7RQKHfMVCwgQHwhGMAc
See attached file.
5- http://www.eolss.net/sample-chapters/c03/e6-54-10-03.pdf
See attached paper.
Hoping this will be helpful,
Rafik
to detect the gene, you can simply use the PCR and sequencing of nifH gene
to detect the activity of Nitrogenase, you can use either ARA Acetylene Reduction Assay , using Gas chromatography
or to use the RT-PCR to measure the gene expression
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