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Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*
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
PREPARATION OF WORKING SOLUTION
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 EXPERIMENTAL PROTOCOL
Sample Protocol for detection of His-tagged Protein in PVDF
- Prepare your PVDF blots as needed (block nonspecific binding sites and wash the blot).
- Incubate the blot with 20 mL of Staining Solution at room temperature for 30 minutes.
- Wash the blots, and pursuit for chemiluminescence detection.
Sample Protocol for Biotin-X NTA–Probed Blot Stripping
- 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. - Wash the blot in Wash Buffer at room temperature 2-4 times for 5 minutes each and proceed with antibody detection.
Calculators
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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 139.669 µL | 698.344 µL | 1.397 mL | 6.983 mL | 13.967 mL |
| 5 mM | 27.934 µL | 139.669 µL | 279.337 µL | 1.397 mL | 2.793 mL |
| 10 mM | 13.967 µL | 69.834 µL | 139.669 µL | 698.344 µL | 1.397 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Citations
View all 4 citations: Citation Explorer
Malaria Detection with Cyclic Catch-and-Release Signal Amplification
Authors: Jacobs, Michael Zachary
Journal: (2017)
Authors: Jacobs, Michael Zachary
Journal: (2017)
A low-cost, high-performance system for fluorescence lateral flow assays
Authors: Lee, Linda G and Nordman, Eric S and Johnson, Martin D and Oldham, Mark F
Journal: Biosensors (2013): 360--373
Authors: Lee, Linda G and Nordman, Eric S and Johnson, Martin D and Oldham, Mark F
Journal: Biosensors (2013): 360--373
Implementation of P22 viral capsids as nanoplatforms
Authors: Kang, Sebyung and Uchida, Masaki and O'Neil, Alison and Li, Rui and Prevelige, Peter E and Douglas, Trevor
Journal: Biomacromolecules (2010): 2804--2809
Authors: Kang, Sebyung and Uchida, Masaki and O'Neil, Alison and Li, Rui and Prevelige, Peter E and Douglas, Trevor
Journal: Biomacromolecules (2010): 2804--2809
Calcium-dependent activation of transglutaminase 2 by nanosecond pulsed electric fields
Authors: Morotomi-Yano, Keiko and Yano, Ken-ichi
Journal: FEBS Open Bio
Authors: Morotomi-Yano, Keiko and Yano, Ken-ichi
Journal: FEBS Open Bio
References
View all 78 references: Citation Explorer
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)
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
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
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
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
Authors: McKay BE, Molineux ML, Turner RW.
Journal: J Comp Neurol (2004): 86
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