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CTAB-PAGE Richard J. Simpson Cold Spring Harb Protoc; doi: 10.1101/pdb.prot5412 Email Alerting Service Subject Categories
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Protocol
CTAB-PAGE Richard J. Simpson INTRODUCTION Although SDS-PAGE is the method of choice for most denaturing gel electrophoresis procedures, the anionic detergent SDS still presents some drawbacks. For example, SDS forms crystals at low temperatures and, in some cases, causes proteins to aggregate or precipitate. In addition, some proteins are not well-resolved in SDS gels or may migrate anomalously. In these situations, the use of a cationic detergent for PAGE offers an alternative approach. The system described in this protocol uses the cationic detergent cetyltrimethyl ammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine (used as a stacking agent) and tricine (N-tris[hydroxymethyl]-methylglycine) used as a counterion and buffer. Some proteins separated on the CTAB electrophoresis system retain their native enzymatic activity, provided the samples are prepared without boiling and without the addition of a reducing agent.
RELATED INFORMATION This protocol is a variation on the standard SDS-PAGE method described in SDS-PAGE of Proteins (Simpson 2006). The discontinuous, cationic detergent PAGE method described here is based on the method of Atkins et al. (1992).
MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and
recipes for reagents marked with .
Reagents All reagents should be electrophoresis grade or better.
-Mercaptoethanol (2%) (optional; see Step 3) CTAB sample buffer Protein samples, lyophilized or as pellets Resolving gel mixture Stacking gel mixture Tricine running buffer (5X)
Equipment Boiling water bath (optional; see Step 3) Micropipettor and tips
Adapted from Proteins and Proteomics by Richard J. Simpson. CSHL Press, Cold Spring Harbor, NY, USA, 2003. Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5412 © 2010 Cold Spring Harbor Laboratory Press
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METHOD 1. Prepare the gels as described in SDS-PAGE of Proteins (Simpson 2006), but use the resolving gel
mixture and stacking gel mixture described in this protocol. 2. Fill the upper and lower buffer reservoirs with 1X tricine running buffer. 3. Prepare the protein samples by dissolving them in 50 µL of CTAB sample buffer at room temperature,
to a final concentration of 5 mg/mL. If retaining biological activity is not a concern, heat the samples for 3 min in a boiling water bath in the presence of 2% -mercaptoethanol. 4. Load the samples into the sample wells. 5. Follow the basic procedure for electrophoresis in SDS-PAGE of Proteins (Simpson 2006), with the
specific details described here and in Steps 6 and 7. Start electrophoresis from the anode (+) to the cathode (−) at 100 V as the samples migrate through the stacking gel. 6. Increase the voltage to 150 V as the samples begin to migrate through the resolving gel. 7. Turn off the power supply and stop the electrophoresis when the dye front reaches the bottom of
the gel.
REFERENCES 172–178. Simpson RJ. 2006. SDS-PAGE of proteins. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4313.
Atkins RE, Levin PM, Taun RS. 1992. Cetyltrimethyl-ammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity. Anal Biochem 202:
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