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Aptamer Best Practices: Dot 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
Like Western Blot1,2 a dot blot is an analytical technique uses to detect proteins. A mixture
of 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
Biotinylated aptamers
PVDF membrane
100% Methanol (MeOH)
1X Phosphate-buffered saline (PBS), pH= 7.4
1% of Non-fat dry milk (NFDM)
Wash solution:1X PBS and 1 mM MgCl2, pH= 7.4
Streptavidin-Alkaline Phosphatase (Sigma #S2890)
ELF®97 Phosphatase Substrate (Invitrogen #E6588)
Blacklight blue” light source (GE bulb F15T8 BLB 15W)
Membrane preparation:
Prewet the PVDF membrane with 100% Methanol (MeOH) then allow to dry. Spot 0.5-2 µL of protein on
the PVDF membrane. Air dry the PVDF for 15 min at room temperature. Wash 2 times with 1X Phosphate-buffered saline (PBS) for 5 min each. Block the membrane with 1% of Non-fat dry milk (NFDM) for
10 mins and then wash 2 times with 1X PBS for 5 mins.
Protein:Aptamer Binding and Washing:
All aptamers should be resuspended to 100uM pH = 7.5 10 mM Tris-HCl and 0.1 mM EDTA.
**Aptamers need to be heated to 85-90C for 2 minutes and then cooled to room temperature prior to use**
9307 W. Broadway, Suite 390, Pearland, TX 77584 • basepairbio.com • [email protected]
Aptamer Best Practices: Dot Blot
Protein:Aptamer Binding and Washing:
Add folded biotin-modified aptamer in a solution of 1X PBS and 1 mM MgCl2 to the immobilized protein.
Incubate at room temperature for 30 min. Wash 2 times with wash solution for 5 min each. Add Streptavidin – AP diluted 1:2000 in wash buffer for 30 mins. Wash 2 times with wash solution for 5 min each.
Add ELF®97 Phosphatase Substrate diluted 1:2000 in wash buffer and incubate for 30 mins. Wash 2
times with wash solution for 5 min each. Excite and image PVDF membrane with a long wavelength
“blacklight blue” source
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]