1.8 nm Ni-NTA-Nanogold^®
[1.8 nm Ni-NTA-Nanogold® product page]

Technical Assistance Online
  Instructions (PDF)
  Material Safety Data Sheet (PDF)

Table of Contents

• Introduction
• Product Information
• General Considerations for the Use of Ni-NTA-Nanogold^®
• Special Considerations for Direct Viewing of Ni-NTA-Nanogold^® in the
• Electron Microscope
• Gold or Silver Enhancement of Ni-NTA-Nanogold^® for EM
• Blotting Applications
• References and Most Recent Publications of Using Ni-NTA-Nanogold^®

Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Non radioactive and non carcinogenic.

Introduction

Contents

Figure 1: Interaction between a His-tagged protein and Ni-NTA-Nanogold^®.

Product Information

General Considerations for the Use of Ni-NTA-Nanogold^®

1. For best results, prepare protein sample in a binding buffer of 20 mM Tris, 0.15 M NaCl, pH 7.6 with 0.1% (w/v) Tween^® -20 and 1% (w/v) nonfat dried milk. Including Tween^® -20 and nonfat dried milk helps reduce the non-specific binding of Ni-NTA-Nanogold^® to the proteins. If significant amounts of non-specific staining are observed at the end of the detection, the concentration of sodium chloride in the binding buffer can be increased to 0.5 to 1 M.

2. The binding buffer must be free from thiols such as beta-mercaptoethanol, or reducing or chelating agents such as DTT, EDTA or citrate. Samples containing EDTA, DTT, or citrate may give low specific staining.

3. Add required amount of Ni-NTA-Nanogold^®, diluted 1/5 1/100 in 20 mM Tris, 150 mM NaCl, 1% nonfat dried milk and 0.1% Tween 20 at pH 7.6, to the His-tagged proteins in the binding buffer. Incubate for 5 -30 minutes at room temperature. The optimum quantity of Ni-NTA-Nanogold^® to be used needs to be determined for each application. Using five to ten fold molar excess of Ni-NTA-Nanogold^® can help reduce the formation of cross-linking products. For most applications, 5-10 minute incubation is sufficient for obtaining a positive staining.

4. After incubation, the excess Ni-NTA-Nanogold^® can be removed by size exclusion liquid chromatography, dialysis or simply washed off from cell or tissue samples or membranes using a buffer containing 50 mM Tris, 300 mM NaCl, 10-200 mM imidazole and 0.1% Tween 20 adjusted to pH 7.6. The wash buffer may be optimized for specific applications by adjusting the imidazole and NaCl (up to 1M) concentration. Increasing the imidazole concentration helps reduce the non-specific interaction or background, and can also decease the binding to the target proteins. To determine the optimal combination of specific staining and background, different wash conditions should be tested.

5. Buffers other than the suggested ones can be used if similar pH and ionic strength are used. In low pH (<5.0) buffers, histidines will be protonated, which disrupts their interaction with the metal.

6. The Ni-NTA procedure can also be used under denaturing conditions. This can be useful in cases where the His-tag is inaccessible from the surface of the fusion proteins or if the protein is insoluble.

7. For the application involving tissues or cells, block the sample with 20 mM Tris, 150 mM NaCl, 0.1% Tween 20 at pH 7.6 containing 1-5% Nonfat dried milk for 5-30 minutes at room temperature prior to the application of Ni-NTA-Nanogold^®. The blocking step can block some non-specific protein binding sites and minimizes non-specific interaction.

8. For most work, gold or silver enhancement with a neutral pH is recommended to give an enhanced signal for detection. The enhancement can be terminated by immersion of the sample or grid in deionized water or 2.5% of sodium thiosulfate for 1 minute as soon as satisfactory specific staining is reached. Further enhancement may lead to undesirable background. Applying gold or silver enhancement to a control sample without exposing to Ni-NTA-Nanogold^® helps to determine the stability time when self-nucleation of the enhancement starts. Self-nucleation can generate high background. The actual enhancement time should never exceed the stability time.

9. Because the 1.8 nm Nanogold^® particles are small, over-staining with OsO_{4, uranyl acetate or lead citrate may tend to obscure direct visualization of individual Nanogold^® particles. Therefore, the use of reduced amount or concentration of usual stains is recommended.}

Contents

Special Considerations for Direct Viewing of 1.8 nm Ni-NTA-Nanogold^® in the Electron Microscope

Nanogold^® provides a much improved resolution and smaller probe size over other colloidal gold antibody products. However, because Nanogold^® is only 1.8 nm in diameter, it will not only be smaller, but will appear less intense than, for example, a 5 nm gold particle. With careful work, however, Nanogold^® may be seen directly through the binoculars of a standard EM even in 80 nm thin sections. However, achieving the high resolution necessary for this work may require new demands on your equipment and technique. Several suggestions follow:

1. Before you start a project with Nanogold^® it is helpful to see it so you know what to look for. Dilute the Nanogold^® stock 1:5 and apply 4 µL to a grid for 1 minute. Wick the drop and wash with deionized water 4 times.

2. View Nanogold^® at 100,000 X magnification with 10 X binoculars for a final magnification of 1,000,000 X. Turn the emission up full and adjust the condenser for maximum illumination.

3. The alignment of the microscope should be in order to give 0.3 nm resolution. Although the scope should be well aligned, you may be able to skip this step if you do step 4.

4. Objective stigmators must be optimally set at 100,000 X. Even if the rest of the microscope optics is not perfectly aligned, adjustment of the objective stigmators may compensate and give the required resolution. You may want to follow your local protocol for this alignment but since it is important, a brief protocol is given here:
   1. At 100,000 X (1 X 10⁶ with binoculars), over focus, under focus, then set the objective lens to in focus. This is where there is the least amount of detail seen.

   2. Adjust each objective stigmator to give the least amount of detail in the image.

   3. Repeat steps a and b until the in focus image contains virtually no contrast, no wormy details, and gives a flat featureless image.

5. Now underfocus slightly, move to a fresh area, and you should see small black dots of 1.8 nm size. This is the 1.8 nm Ni-NTA-Nanogold^®. For the 1:5 dilution suggested, there should be about 5 to 10 gold spots on the small viewing screen used with the binoculars. Contrast and visibility of the gold clusters is best at 0.2 - 0.5 m defocus, and is much worse at typical defocus values of 1.5 - 2.0 m commonly used for protein molecular imaging.

6. In order to operate at high magnification with high beam current, thin carbon film over fenestrated holey film is recommended. Alternatively, thin carbon or 0.2% Formvar over a 1000 mesh grid is acceptable. Many plastic supports are unstable under these conditions of high magnification/high beam current and carbon is therefore preferred. Contrast is best using thinner films and thinner sections.

7. Once you have seen 1.8 nm Ni-NTA-Nanogold^® you may now be able to reduce the beam current and obtain better images on film. For direct viewing with the binoculars reduction in magnification from 1,000,000 X to 50,000 X makes the Nanogold^® much more difficult to observe and not all of the golds are discernable. At 30,000 X (300,000 X with 10 X binoculars) Nanogold^® particles are not visible. It is recommended to view at 1,000,000 X, with maximum beam current, align the objective stigmators, and then move to a fresh area, reduce the beam, and record on film.

8. If the demands of high resolution are too taxing or your sample has an interfering stain, a very good result may be obtained using gold or silver enhancement to give particles easily seen at lower magnification.

Contents

Gold or Silver Enhancement of 1.8 nm Ni-NTA-Nanogold^® for EM

Protein samples or pecimens must be thoroughly rinsed with deionized water before silver enhancement reagents are applied. This is because the buffers used may contain chloride ions and other anions which form insoluble precipitates with silver. These are often light-sensitive and will give non-specific staining.

Fixing with osmium tetroxide may cause some loss of silver; if this is found to be a problem, slightly longer development time may be appropriate. Alternatively, use of 0.1% osmium tetroxide instead of 1% has been found to give similar levels of staining while greatly reducing etching of the silver particles.

Contents

Blotting Applications

1. Spot or transfer his-tagged proteins onto PVDF or nitrocellulose membrane.
2. Equilibrate membranes with 20 mM Tris, 150 mM NaCl, pH 7.6 and 0.1% (w/v) Tween^® 20 (TBST) for 2 min.
3. Block with 20 mM Tris, 150 mM NaCl, pH 7.6 containing 5% nonfat dried milk and 0.1 % Tween^® 20 for 30 minutes at room temperature
4. Rinse with TBST (3 X 5 mins).
5. Incubate with 1.8 nm Ni-NTA-Nanogold^® diluted 1 : 50 in 20 mM Tris, 150 mM NaCl, pH 7.6 containing 1% nonfat dried milk, and 0.1% Tween^® 20 for 5 minutes at room temperature.
6. Rinse with TBST and 10 mM imidazole (3 X 5 min). The concentration of imidazole and sodium chloride can be increased if high non-specific binding is observed.
7. Rinse with deionized water (4 X 3 mins).
8. Perform gold or silver enhancement as directed in the instructions for gold or silver reagents.
9. Stop the enhancement when satisfactory staining is achieved. The enhancement can be stopped by washing membrane with deionized water (3 X 5 mins).

Contents

References

1. Hochuli, E.; Bannwarth, W.; Döbeli, H.; Gentz, R., and Stber, D.: Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology, 6, 1321-1325 (1988).

2. Porath, J., and Olin, B.: Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry, 22, 1621-1630 (1983).

3. Hochuli, E.; Döbeli, H., and Schacher, A.: New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatograph., 411, 177-184 (1987).

4. Uhlén, M., and Moks, T.: Gene fusions for purpose of expression: an introduction. Methods Enzymol., 185, 129-143 (1990).

5. Casey, J. L.; Keep, P. A.; Chester, K. A.; Robson, L.; Hawkins, R. E., and Begent, R. H. J.: Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography. J. Immunol. Meth., 179, 105-116 (1995).

6. Schmitt, J.; Hess, H., and Stunnenberg, H. G.: Affinity purification of histidine-tagged proteins. Molecular Biology Reports, 18, 223-230 (1993).

7. Kollman, J. M.; Zelter, A.; Muller, E. G.; Fox, B.; Rice, L. M.; Davis, T. N., and Agard, D. A.: The Structure of the gamma-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation. Mol. Biol. Cell, 19, 207-215 (2008).

8. Kollman, J. M.; Zelter, A.; Muller, E. G.; Fox, B.; Rice, L. M.; Davis, T. N., and Agard, D. A.: The Structure of the gamma-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation. Mol. Biol. Cell, 19, 207-215 (2008).

9. Balasingham, S. V.; Collins, R. F.; Assalkhou, R.; Homberset, H.; Frye, S. A.; Derrick, J. P., and Tonjum, T.: Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis. J. Bacteriol., 189, 5716-5727 (2007).

10. Jiang, Z. G.; Simon, M. N.; Wall, J. S., and McKnight, C. J.: Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B. Biophys. J., 92, 4097-4108 (2007).

11. Pye, V. E, Beuron, F, Keetch, C. A, McKeown, C, Robinson, C. V, Meyer, H. H, Zhang, X, and Freemont, P. S.: Structural insights into the p97-Ufd1-Npl4 complex. Proc. Natl. Acad. Sci. USA, 104, 467-472 (2007).

12. Promnares, K.; Komenda, J.; Bumba, L.; Nebesarova, J.; Vacha, F., and Tichy, M.: Cyanobacterial Small Chlorophyll-binding Protein ScpD (HliB) Is Located on the Periphery of Photosystem II in the Vicinity of PsbH and CP47 Subunits. J. Biol. Chem., 281, 32705-32713 (2006).

13. Collins, R. F.; Beis, K.; Clarke, B. R.; Ford, R. C.; Hulley, M.; Naismith, J. H.; and Whitfield, C.: Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules. J. Biol. Chem., 281, 2144-2150 (2006).

14. Wolfe, C. L.; Warrington, J. A.; Treadwell, L., and Norcum, M. T.: A three-dimensional working model of the multienzyme complex of aminoacyl-tRNA synthetases based on electron microscopic placements of tRNA and proteins. J. Biol. Chem., 280, 38870-38878 (2005).

15. Bumba, L.; Tichy, M.; Dobakova, M.; Komenda, J., and Vacha, F.: Localization of the PsbH subunit in photosystem II from the Synechocystis 6803 using the His-tagged NiNTA Nanogold labeling. J. Struct. Biol., 152, 28-35 (2005).

16. Collins, R. F.; Frye, S. A.; Balasingham, S.; Ford, R. C.; Tonjum, T., and Derrick, J. P.: Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J. Biol. Chem., 280, 18923-18930 (2005).

17. Collins, R. F.; Frye, S. A.; Balasingham, S.; Ford, R. C.; Tonjum, T., and Derrick, J. P.: Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J. Biol. Chem., 280, 18923-18930 (2005).

18. Chatterji, A.; Ochoa, W. F.; Ueno, T.; Lin T., and Johnson, J. E.: A virus-based nanoblock with tunable electrostatic properties. Nano Lett., 5, 597-602 (2005).

19. Hainfeld, J. F.; Liu, W.; Joshi, V., and Powell, R. D.: Nickel-NTA-Nanogold Binds his-Tagged Proteins. Microsc. Microanal., 8, (Suppl. 2: Proceedings); Voekl, E.; Piston, D.; Gauvin, R.; Lockley, A. J.; Bailey, G. W., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 832CD (2002) (Paper).

20. Buchel, C.; Morris, E.; Orlova, E., and Barber, J.: Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis. J. Mol. Biol., 312, 371-379 (2001).

21. Hainfeld, J. F.; Liu, W.; Halsey, C. M. R.; Freimuth, P., and Powell, R. D.: Ni-NTA-Gold Clusters Target His-Tagged Proteins. J. Struct. Biol., 127, 185-198 (1999).

Technical Assistance Available.

For a complete list of references citing this product, please visit our References page.

Contents