8 M Guanidine Hydrochloride Solution

Buffered, pH 8.5

Product Code G 7294

Store at Room Temperature

Product Description Precautions and Disclaimer

This product is a ready-t o-use 8 M guanidine This product is for R&D use only, not for drug,
hydrochloride solution buffered at pH 8.5 with 0.05 M household, or other uses. Please consult the Material
bicine. It is ideal for use with affinity tagging procedures Safety Data Sheet for information regarding hazards
such as labeling and modification of cysteine residues. and safe handling practices.
The bicine buffer does not contain primary amines,
phosphates, or carboxyl groups, and therefore, is Preparation Instructions
compatible with mass spectrometric procedures. The product is a clear, colorless liquid and is supplied
ready-to-use. The 8 M Guanidine Hydrochloride
Guanidine hydrochloride is commonly used as a Solution may be used as a stock solution to prepare
denaturant, because of its ability to break hydrogen solutions of lower concentrations as appropriate (see
bonds between amino acid residues. By breaking Table 1).
these bonds, the 3D conformation of the protein is
unfolded and the aqueous solubility of the protein is Table 1.
greatly increased. Once denatured, the protein can be Dilution Table - These dilutions are based on a 10 ml
easily reduced, modified, or analyzed, in a variety of starting volume of the 8 M Guanidine-HCl buffered
procedures. Further processing of the sample can solution.
allow the proteins to be analyzed by other common
protein analysis methods including mass spectrometry, Volume of water to
electrophoresis, and enzymatic digests. Desired
Add to 10 ml of Final Volume
Molarity
8 M Guanidine-HCl
Guanidine hydrochloride has typically been used for the 8 M 0.0 ml 10.0 ml
isolation of RNA, to denature globular proteins, and for 7 M 1.4 ml 11.4 ml
protein refolding studies. It can also be used to
6 M 3.3 ml 13.3 ml
facilitate the generation of tryptic peptides for analysis
5 M 6.0 ml 16.0 ml
of complex protein samples.
4 M 10.0 ml 20.0 ml
3 M 16.7 ml 26.7 ml
Reagents Required But Not Provided For Optional
Procedure 2 M 30.0 ml 40.0 ml
1 M 70.0 ml 80.0 ml
200 mM Tributylphosphine Solution (TBP, Product
Code T 7567, supplied ready-to-use) Storage/Stability
Store the product at room temperature. The product is
0.5 M Iodoacetamide Solution (prepared from Product stable for at least one year in an unopened container.
Code A 3221 and water)

100 mM Ammonium Bicarbonate Solution (prepared

from Product Code A 6141 and water)

0.2 mg/ml Proteomics Grade Trypsin Solution

(prepared from Product Code T 6567 and 1 mM HCl)
2

Procedure 7. Add 100 µl of the 0.2 mg/ml Proteomics Grade

A. Protein Denaturation and Solubilization of Cell Trypsin Solution to the dialyzed sample. (For more
Paste information, see the Technical Bulletin for Product
1. Add 1 ml of the 8 M Guanidine Hydrochloride Code T 6567.)
Solution for every 0.1 gram of wet cell paste. 8. Incubate the sample overnight at 37 °C in a water
2. Vortex the suspension for 2 minutes. bath.
3. Centrifuge the suspension at 15,000 x g for 9. Peptide solutions can now be analyzed by mass
10 minutes at room temperature to remove cell spectrometric methods.
debris.
4. After centrifugation, carefully remove the References
supernatant containing the soluble proteins. 1. Bruice, P.Y., Organic Chemistry, 3rd Ed., Prentice
Note: For SDS-PAGE analysis, the denaturant Hall (Upper Saddle River, New Jersey: 2001), pp
must be removed by dialysis or protein precipitation 950-953.
with trichloroacetic acid (TCA, Product Code 2. Cox, R.A., The Use of Guanidinium Chloride in the
PROT-PR). Isolation of Nucleic Acids, Methods in Enzymology,
12 B, 120-129 (1968).
B. Reduction and Alkylation of Extracted Proteins and 3. Voet, D. et al., Fundamentals of Biochemistry,
Tryptic Digestion (Optional) Upgrade Edition., John Wiley & Sons Inc (New
1. Reduce the protein sample (section A, step 4) by York, New York: 2002), pp 151-152.
adding 25 µl of the 200 mM Tributylphosphine 4. Lodish, et al., Molecular Cell Biology, 4th Ed., W.H.
Solution per 1 ml of protein solution. Incubate the Freeman and Company, (New York, New York:
sample at room temperature for 15-30 minutes. 2000). p 63.
2. Alkylate the protein solution by adding 30 µl of the 5. Gygi, S.P., et al., Quantitative Analysis of Complex
0.5 M Iodoacetamide Solution per 1 ml of protein Protein Mixtures Using Isotope-coded Affinity Tags.
solution. Incubate the sample at room temperature Nature Biotechnology, 17, 994-999 (1999).
for 1 hour. 6. West, S.M., et al., Improved Protein Refolding
3. Quench the excess iodoacetamide with additional Using Hollow-Fibre Membrane Dialysis. Biotechnol.
TBP. Add 25 µl of the 200 mM Tributylphosphine Bioeng., 57, 590-599 (1998).
Solution per 1 ml of protein solution and incubate 7. Lilie, H., et al., Advances in Refolding of Proteins
for 15 minutes at room temperature. Produced in E. Coli. Current Opinion in
4. Centrifuge the sample at 20,000 x g for five minutes Biotechnology, 9, 497-501 (1998).
at room temperature to pellet any insoluble 8. Marston, F.A.O., and Hartley, D.L., Solubilization of
material. Protein Aggregates. Methods Enzymol., 182, 264-
5. Dialyze the sample against 1 liter of 100 mM 276 (1990).
Ammonium Bicarbonate Solution for 1 hour at room
temperature. BE/MAM 5/04
6. Replace the dialysis buffer with fresh 100 mM
Ammonium Bicarbonate Solution and continue to
dialyze for another 2 hours. If a precipitate forms
during dialysis, resuspend the precipitated proteins
by pipetting the solution up and down. Transfer the
solution to a clean tube. The precipitate will clear
upon enzymatic digestion.

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