Alcian Blue Sulfate Quantification Protocol

Dominik Haudenschild, March 2007

from Masuda-Thonar (1994 Anal Biochem)

Procedure 

· Dissolve Agarose in Extraction buffer, or use supernatant neat

· Add 75ul Dilution Buffer to 96-well filter-bottom plate

· Add 25ul Sample

· Add 150ul Alcian Blue Dye

· Agitate 1 hour at R.T.

· Filter through membrane

· Wash 3x200ul with Wash Buffer

· Blot dry (don't dry completely with vacuum)

· Remove rubber seal around bottom of filter plate

· If using Scintillation Fluid to quantify radioactivity:

· Punch out membrane with biopsy punch

· Dissolve 1 hour in 500ul Dissolving buffer

· Add 5ml Scintillation Fluid

· Count

· If using Phosphorimager to quantify radioactivity:

· Dry bottom of filter plate completely

· Erase phosphor-storage screen for 5 minutes

· Expose bottom of filter plate directly to phosphor-storage screen for ?? hours in light-proof container

· Scan image using phosphorimager in core lab

· Quantitate pixel intensity using phosphorimager software

Solutions

· Extraction Buffer for Agarose/Alginate

· 4M Guanidine

· 50mM Sodium Acetate pH 6.0

· Protease inhibitors

· 0.1M 6-aminohexanoic acid

· 10mM EDTA

· 5mM Benzamidine HCl

· 10mM N-EthylMaleimide

· 1mM PMSF

· Dilution Buffer

· 50mM Sodium Acetate pH 5.8

· 0.5% Triton X-100

· Alcian Blue Dye (Keep no more than 3 weeks)

· 0.2% Alcian Blue (Electrophoresis grade)

· 50mM Sodium Acetate pH 5.8

· 85mM MgCl2

· Completely dissolve, then filter through whatman #1 paper or coffee filter

· Wash Buffer

· 50mM Sodium Acetate pH 5.8

· 100mM Sodium Sulfate

· 50mM MgCl2

· Dissolving Buffer

· 4M Guanidine HCl

· 33% Isopropanol

Equipment

· 96-well 0.45uM Hydrophillic PVDF Filter-bottom plates

· Vacuum manifold

· Scintillation Fluid and counter

· Phosphor-storage screen and phosphorimager