GOLDIBLOT PRODUCT INFORMATION

GoldiBlot His Western Blot Kit
[GoldiBlot His Western Blot Kit product page]

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  Instructions (PDF)

Table of Contents

• Intended Use
• Principle of GoldiBlot His Western Blot Kit
• Reagents Supplied
• Materials Required, but not Supplied
• Storage
• Procedure for Detection of Polyhistidine-Tagged Proteins
• References

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.

Intended Use

Principle of GoldiBlot His Western Blot Kit

(left) Ni-NTA-Gold, showing mechanism of binding to a polyhistidine (His) tagged protein. (right) Principle of GoldiBlot: gold binding followed by autometalographic amplification (deposition of metal selectively onto the gold particles) generates visible signal.

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Reagents Supplied

GoldiBlot Nickel-NTA-Gold	1.5 mL
GoldiBlot AutoMet Detect A	40 mL
GoldiBlot AutoMet Detect B	40 mL
GoldiBlot AutoMet Detect C	40 mL
GoldiBlot AutoMet Detect D	40 mL

Materials Required, but not Supplied

1. Tris Buffered Saline Tween®-20 (TBST): 20 mM Tris, 0.15 M NaCl, pH 7.6, 0.1% (w/v) Tween®-20
2. 5% (w/v) nonfat dried milk in TBST
3. Tris Buffered High Salt Saline Tween#174;-20 (TBST-High NaCl): 20 mM Tris, 1.5 M NaCl, pH 7.6, 1% (w/v) Tween®-20
4. 1% (w/v) nonfat dried milk in TBST-High NaCl
5. 10 mM imidazole in TBST-High NaCl

Storage

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Procedure for Detection of Polyhistidine-Tagged Proteins

All GoldiBlot reagents and other required materials should be equilibrated to room temperature prior to the blotting procedure. All incubations of the Goldiblot western blotting should be performed at room temperature with gentle shaking, preferably using an orbital shaker to ensure that the membrane remains immersed.

1. Transfer protein from gel to a PVDF membrane.
   Note: Although other membranes can be used, optimization may be required.
2. Place the membrane in a tray and equilibrate with TBST for 2 minutes.
3. Block the membrane with 5% (w/v) nonfat dry milk in TBST for 12 minutes.
4. Place the membrane in 10 mL of 1% (w/v) nonfat dried milk in TBST-High NaCl. Add 0.1 mL of GoldiBlot Nickel-NTA-Au to the 10 mL of Milk/TBST-High NaCl. Incubate the blot for 20 minutes.
5. Wash the membrane three times with 15 mL of 10 mM imidazole in TBST-High NaCl for 3 minutes each.
6. Wash the membrane three times with 15 mL of deionized water for 3 minutes each.
7. Before starting the last deionized water wash, mix 2.5 mL GoldiBlot AutoMet Detect A with 2.5 mL B in a clean 15 mL container. After 5 minutes, add 2.5 mL C and 2.5 mL D to the mixture of A and B, and mix. Incubate the blot with 10 mL of the ABCD mixture for 6 to 15 minutes, or until satisfactory staining is reached.
   Note: The incubation time of GoldiBlot AutoMet Detect ABCD depends on the quantities of His-tagged proteins loaded. The bands loaded with more than 100 ng His-tagged proteins can be seen within 6 minutes. Longer incubation time may be needed in order to see less than 20 ng His-tagged proteins. However, longer incubation may lead to the visualization of some non specific background bindings.
8. Wash the membrane three times with 15 mL of deionized water for 3 minutes each to terminate the autometallographic amplification.
   Note: any light purple color membrane background will fade away as the membrane dries out.
9. Air-dry the membrane.

Note: The concentration of NaCl and Tween 20 in TBST-High NaCl (used in GoldiBlot Nickel-NTA-Au binding and imidazole washes) can be slightly adjusted to achieve an optimized signal-to-noise ratio. Less NaCl and Tween-20 can enhance the band intensity of His-tagged proteins, and higher NaCl and Tween-20 help reduce the non specific background staining.

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References

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

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

3. 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).

4. 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).

5. 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).

6. 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).

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

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