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Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*

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Physical properties
Molecular weight715.98
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Desiccated
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Molecular weight
Biotin-X nitrilotriacetic acid (biotin-X NTA) is widely used to detect histidine-tagged proteins immobilized on nitrocellulose membranes. The NTA moiety of biotin-X NTA chelates Ni ion that is also chelated with histidine tags. The NTA-polyHis-complex can be detected using standard enzyme-linked streptavidin methods. Biotin-X NTA can be used to detect less than 0.1 pmol of histidine-tagged protein using a streptavidin'horseradish peroxidase conjugate and chemiluminescence techniques. Biotin-X NTA can be removed from the histidine-tagged protein at pH 4.8, allowing the blot to be reanalyzed with another probe. In combination with fluorescent avidin conjugates, this NTA biotin derivative can be used for detecting polyhistidine-containing biomolecules such as fusion proteins. This NTA Biotin derivative is a bifunctional reagent that is used to detect histidine-tagged proteins immobilized. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.

Example protocol


1. Biotin-X NTA working solution (1mg/mL):
Add 1 mL of DMSO or ddH2O to Biotin-X NTA vial and mix well.

2. Staining Solution:
Prepare fresh Staining Solution (less than 30 minutes before use): prepare 20 mL of Staining Solution for each 8 cm ×10 cm blot by adding 20 μL of 10 mM NiCl2 , 20 μL of 1 mg/mL biotin-X NTA working solution and 1-2 μL of 1 mg/mL streptavidin–alkaline phosphatase to 20 mL of Blocking Buffer. Mix well. Note: Once the solution is made, it is ready to use right away, and remains stable for at least 30 minutes.


Sample Protocol for detection of His-tagged  Protein in PVDF

  1. Prepare your PVDF blots as needed (block nonspecific binding sites and wash the blot).

  2. Incubate the blot with 20 mL of Staining Solution at room temperature for 30 minutes.

  3. Wash the blots, and pursuit for chemiluminescence detection.

Sample Protocol for Biotin-X NTA–Probed Blot Stripping

  1. If desired, the blot can be stained using other detection methods.
    For staining with antibodies or lectins, strip the biotin-X NTA complex off the blot by incubating it in 62.5 mM Tris, 0.2% SDS, 50 mM dithiothreitol (DTT), pH 6.8, at 50°C for 40 minutes with gentle agitation. Or incubated overnight in 200 mM acetate acid with 40 mM EDTA, pH 4.8.

  2. Wash the blot in Wash Buffer at room temperature 2-4 times for 5 minutes each and proceed with antibody detection.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM139.669 µL698.344 µL1.397 mL6.983 mL13.967 mL
5 mM27.934 µL139.669 µL279.337 µL1.397 mL2.793 mL
10 mM13.967 µL69.834 µL139.669 µL698.344 µL1.397 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles



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Evaluation of a new biotin-DOTA conjugate for pretargeted antibody-guided radioimmunotherapy (PAGRIT((R)))
Authors: Urbano N, Papi S, Ginanneschi M, De Santis R, Pace S, Lindstedt R, Ferrari L, Choi S, Paganelli G, Chinol M.
Journal: Eur J Nucl Med Mol Imaging. (2006)
A biotin-protein bond with stability in plasma
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2005): 98
Probing the interaction of the biotin-avidin complex with the relaxivity of biotinylated Gd-DTPA
Authors: Langereis S, Kooistra HA, van Genderen MH, Meijer EW.
Journal: Org Biomol Chem (2004): 1271
Immunoassay of total prostate-specific antigen using europium(III) nanoparticle labels and streptavidin-biotin technology
Authors: Huhtinen P, Soukka T, Lovgren T, Harma H.
Journal: J Immunol Methods (2004): 111
Biotin is endogenously expressed in select regions of the rat central nervous system
Authors: McKay BE, Molineux ML, Turner RW.
Journal: J Comp Neurol (2004): 86
Design, synthesis, and evaluation of 5'-S-aminoethyl-N(6)- azidobenzyl-5'-thioadenosine biotin conjugate: a bifunctional photoaffinity probe for the es nucleoside transporter
Authors: Addo JK, Buolamwini JK.
Journal: Bioconjug Chem (2004): 536
Instability of the biotin-protein bond in human plasma
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2004): 156
Volume of RBCs, 24- and 48-hour posttransfusion survivals, and the lifespan of (51)Cr and biotin-X-N-hydroxysuccinimide (NHS)-labeled autologous baboon RBCs: effect of the anticoagulant and blood pH on (51)Cr and biotin-X-NHS elution in vivo
Authors: Valeri CR, Pivacek LE, Cassidy GP, Ragno G.
Journal: Transfusion (2002): 343
Endogenous biotin staining in a subset of spinal neuronal cell bodies: a potential confounding factor for neuroanatomical studies
Authors: Berkowitz A., undefined
Journal: Brain Res (2002): 98
End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads
Authors: Xu H, Zhang S, Liu D, Liang CC.
Journal: Mol Biotechnol (2001): 183