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Nevada Proteomics Center

Phone(775) 784-6337: 2-D Gels
Phone(775) 784-1590: Mass Spectrometry
Fax(775) 784-1419
Emailquilici@unr.edu
Location School of Medicine
Manville Medical Building, Rooms 15-17, MS 0330
Address 1664 N. Virginia Street
Reno,  NV  89557-MS 0330
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Protocol: Choice of Rehydration Buffers for 2-D Electrophoresis

Homemade Rehydration Buffers

Preparation of 8.5 M Deionized Urea

255.2 g urea (Bio-Rad, catalog no. 161-0731) is added to about 250 ml water and stirred until dissolved. To the mixture is added 2.5 g AG 501-X8 resin (Bio-Rad, catalog number 142-6424); the mixture is stirred for about 15-20 minutes and then the solution is filtered or carefully poured off. The final volume is adjusted to 500 ml with water. The solution is aliquoted into plastic bottles or tubes (do not use glass) and stored at -20°C.

Rehydration Buffer for Soluble Proteins (8 M Urea, 4% CHAPS, 10 mM Tris)

Combine the following:
94.1 ml 8.5 M Deionized Urea
4 g CHAPS
0.121 g Tris base
Adjust final volume to 100 ml with water. Aliquot into plastic tubes and store at -20°C.

General Purpose Rehydration Buffer for Soluble Proteins and Loosely Held Membrane Proteins (7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM Tris)

Combine the following:
82.4 ml 8.5 M Deionized Urea
14.0 g Thiourea
4 g CHAPS
0.121 g Tris base
Adjust final volume to 100 ml with water. Aliquot into plastic tubes and store at -20°C.

Rehydration Buffer for Membrane Proteins 1 (5 M Urea, 2 M Thiourea, 2% CHAPS, 2% SB 3-10, 10 mM Tris)

Combine the following:
58.8 ml 8.5 M Deionized Urea
14.0 g Thiourea
2 g CHAPS
2 g SB 3-10
0.121 g Tris base
Adjust final volume to 100 ml with water. Aliquot into plastic tubes and store at -20°C.

Rehydration Buffer for Membrane Proteins 2 (7 M Urea, 2 M Thiourea, 2% ASB-14, 10 mM Tris)

Combine the following:
82.4 ml 8.5 M Deionized Urea
14.0 g Thiourea
2 g ASB-14 (amidosulfobetaine 14)
0.121 g Tris base
Adjust final volume to 100 ml with water. Aliquot into plastic tubes and store at -20°C.

Homemade DIGE Buffers

DIGE Reaction Buffer

Combine the following:
41.25 ml 8.5 M Deionized Urea
7.613 g Thiourea
2 g CHAPS
0.1817 g Tris base
The pH of the mixture is adjusted to 8.72 with diluted HCl. The final volume is adjusted to 50 ml with water.

DIGE Dilution Buffer

Combine the following:
41.25 ml 8.5 M Deionized Urea
7.613 g Thiourea
2 g CHAPS
The final volume is adjusted to 50 ml with water.

Commercially Available Rehydration Buffers

DeStreak Rehydration Solution (GE Healthcare, catalog no.17-6003-19)
This general purpose solution, which contains urea (probably 7M), 2 M thiourea, 4% CHAPS, bromophenol blue and the disulfide exchange reagent DeStreak, is a wonderful rehydration buffer for most applications.

Bio-Rad Sequential Extraction Reagent 2 (Bio-Rad, catalog no.163-2103)
This solution contains 8 M urea, 4% CHAPS, 40 mM Tris and 0.2% 3/10 ampholytes. This solution is excellent if only soluble proteins are desired.

Bio-Rad Sequential Extraction Reagent 3 (Bio-Rad, catalog no. 163-2104)
This solution contains 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 40 mM Tris and 0.2% 3/10 ampholytes. The SB 3-10 in this mixture is a stronger detergent than CHAPS and helps solubilize intrinsic membrane proteins. This rehydration buffer is recommended when the sample contains membrane proteins.

Preparation of Final Rehydration Mixtures

Samples should be prepared in one of the above rehydration buffers. Ideally, extracts used for 11 cm IPG strips should contain 200 – 250 µg total protein per 200 µl. Extracts prepared for use with 24 cm strips may contain 500 – 1000 µg protein per 450 µl, although, for use with DIGE gels, 150 – 250 µg total protein is optimal. If the sample is expected to contain very few types of proteins, much less total protein may be loaded.

Just before rehydrating IPG strips prepared in one of the homemade buffers, the following should be added to the sample:

  • 0.5 µl/100 µl ampholyte solution (usually a 3/10 ampholyte solution, but the ampholyte solution used may vary with the pH range of the IPG strip used)
  • 1.25 µl/100 µl 0.1% bromophenol blue in water
  • 1.2 µl/100 µl DeStreak Reagent (GE Healthcare, catalog no. 17-6003-18; cut the tip off of a pipette tip in order to pipette this viscous solution); Note: 50 mM DTT may be used in place of DeStreak Reagent

If using one of the commercially available rehydration buffers (see above), add whichever of the 3 additions above are not already included in the buffer.

After centrifuging at 16,000 x g for 10 minutes, the supernatant is ready for rehydration of an IPG strip.

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University of Nevada, Reno
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Reno,  NV  89557-

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