BP WB - Base Pair Biotechnologies

Aptamer Best Practices: Western Blot Base Pair Biotechnologies provides custom aptamer development services and catalog aptamers to academic, commercial, and government researchers for a variety of applications. To support their efforts we provide this series of aptamer best practices as a introduction to their use. Additional assistance is provided as needed.

Tertiary structure of a DNA aptamer developed by Base Pair Biotechnologies

Western Blot1,2 is an analytical technique uses to detect proteins. After separation by gel electrophoresis the proteins are transferred to a membrane where they are typically stained with antibodies specific to the target protein, with a secondary antibody used for detection. Aptamers can be substituted in a single step method outlined below Materials and Methods Two-dimensional electrophoresis can be performed according to the carrier ampholine method of isoelectric focusing (O'Farrell, P.H., J. Biol. Chem. 250: 4007-4021, 1975, Burgess-Cassler, A., Johansen, J., Santek, D., Ide J., and Kendrick N., Clin. Chem. 35: 2297, 1989) by Kendrick Labs, Inc. (Madison, WI). Isoelectric focusing can be carried out in a glass tube of inner diameter 2.3 mm using 2.0% pH 3-10 Isodalt Servalytes (Serva, Heidelberg, Germany) for 9,600 volt-hrs. One ug (PVDF) of an IEF internal standard, such as tropomyosin, should be added to each sample. This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and pI 5.2. The enclosed tube gel pH gradient plot should be determined with a surface pH electrode.

After equilibration for 10 min in buffer "O" (10% glycerol, 50mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8), each tube gel should be sealed to the top of a stacking gel that overlaid a 12% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis can be carried out for about 4 hrs at 15 mA/gel. After slab gel electrophoresis, the gels should be placed in transfer buffer (10mMCaps, pH 11.0, 10% MeoH) and transblotted onto a PVDF membrane overnight at 200 mA and approximately 100 volts/ two gels. The following proteins (Sigma Chemical Co., St. Louis, MO) can be used as molecular weight standards: myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000) carbonic anhydrase (29,000) and lysozyme (14,000). These standards should appear at the basic edge of the Coomassie Brilliant Blue R-250-stained membrane.

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Aptamer Best Practices: Western Blot Western Blot Methods The biotinylated aptamers can be diluted to 100 µM in 10 mM Tris pH 7.5, 0.1 mM EDTA. The aptamers should then diluted be 1:5,000,000 in Tween-20 tris buffer saline containing 1mM MgCl2 (TTBS-MG). **Aptamers need to be heated to 85-90C for 2 minutes and then cooled to room temperature prior to use** For first time use a lower dilution can be used.

The blots are stained with Coomassie Brilliant Blue R 250 and desktop scanned. The blots are then blocked for two hours in 5% bovine serum albumin (BSA) in (TTBS-MG). The blots can be incubated for two hours in the aptamers as indicated above and rinsed 3 x 10 minutes in TTBS-MG. Blots are then placed in poly HRP Streptavidin (Thermo, Cat # N200, Lot# OI190258) diluted 1:2,000 in 2% BSA TTBSMG for two hours, rinsed as above, treated with ECL, and exposed to x-ray film.

For additional assistance contact us at [email protected] or call us at 281.829.8876 Custom Services information can be found at www.basepairbio.com Catalog Aptamers can be found at www.aptamersthatwork.com References: 1. 2.

Towbin H., Staehelin T., Gordon J. (1979) Electophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 76 (9); 4350-54. Burnette WN.. (1981) Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry. 112 (2) 195-203.

9307 W. Broadway, Suite 390, Pearland, TX 77584 • basepairbio.com • [email protected]