Supplementary Information
SiaFind™ Pan-Specific Lectenz® 2.0 has a molecular mass of about 57 kD and works as a monomer without bivalent metal ions. It is 8xHis-tagged at its N-terminus, and an anti-polyhistidine antibody, or in the case of the biotinylated version, a streptavidin conjugate can be used for detection. The 8xHis tag may be removed by FasTEV™ (Cat #GE0501), a TEV protease with enhanced stability and catalytic activity.
The original SiaFind™ Pan-Specific Lectenz® has a molecular mass of about 77 kD and works as a monomer without bivalent metal ions. It is 6xHis-tagged at its N-terminus, and an anti-polyhistidine antibody, or in the case of the biotinylated version, a streptavidin conjugate can be used for detection. The fluorescent version can be directly detected.
Form and Storage
The SiaFind™ Pan-Specific Lectenz® 2.0 are supplied lyophilized in a storage buffer (50 mM EPPS, 200 mM NaCl, pH 7.5) and should be reconstituted in 100 μL molecular grade water to yield a 10 mg/mL solution. Concentration is determined by spectrophotometry using E1% 10.4.
The original SiaFind™ Pan-Specific Lectenz® (native and biotinylated) are supplied lyophilized in a storage buffer (50 mM EPPS, 100 mM NaCl, pH 7.5) and should be reconstituted in 100 μL molecular grade water to yield a 10 mg/mL solution. Concentration is determined by spectrophotometry using E1% 12.7.
The SiaFind™ Pan-Specific Lectenz® SureLight® 488 reagent is supplied as a 10 mg/mL solution in a storage buffer (50 mM EPPS, 100 mM NaCl, pH 7.5). Concentration is determined by spectrophotometry using E1% 12.7. The actual absorbance at 280 nm is calculated by measuring the observed absorbance at 280 nm and subtracting the absorbance at 493 nm multiplied by a correction factor of 0.11; A280actual = A280observed – (A493 x 0.11). Store in dark tubes, and perform experiments protected from light.
Once reconstituted, store at 4°C for up to 5 days or -20°C for up to 6 months. Aliquoting is recommended to avoid repeated freeze-thaw cycles.
All 5X buffers should be diluted to 1X with ultrapure water. For instance, to make 250 mL, add 50 mL of any 5X buffer to 200 mL water and mix by inversion. All buffers may be stored at 4 to 25°C.