General Western Blot Protocol:
- Glycoprotein sample size: 500ng
- Lectin Concentration: 0.1ug/ml
- Load samples at 500 ng of glycoprotein per lane
- Run 4-20% Bis-Tris SDS page gel
- Transfer gel to a PVDF membrane
- Block membrane for 1 hr at RT with RIPA buffer (R0278 Sigma)
- Incubate HRP lectin at 0.1ug/ml with RIPA buffer for 2 hours at RT
- Wash membrane 5 x 5 minutes with 25ml RIPA buffer
- Detect using chemiluminescent substrate (CPS1-120)
Detects glycoproteins containing α-D-mannose, α-D-glucose when used with appropriate peroxidase substrate
Con A is not blood group specific but has an affinity for terminal α-D-mannosyl and α-D-glucosyl residues. Ca2+ and Mn2+ ions are required for activity. Con A dissociates into dimers at pH 5.6 or below. Between pH 5.8 and pH 7.0, Con A exists as a tetramer; above pH 7.0 higher aggregates are formed. Con A exhibits mitogenic activity which is dependent on its degree of aggregation. Succinylation results in an active dimeric form which remains a dimer above pH 5.6.
Package size based on protein content
One unit will form 1 mg purpurogallin in 20 sec from pyrogallol at pH 6.0 at 20 °C.
Lyophilized powder containing tris-citrate buffer salts and trace calcium and manganese
Prepared from peroxidase type VI (P8375), using the method of Wilson and Nakane, which promotes conjugation but prevents the interaction between Con A and peroxidase sugar residues.
Where reported, agglutination activity is expressed in μg/ml and is determined from serial dilutions in phosphate buffered saline, pH 6.8, containing Ca2+ and Mn2+ of a 1 mg per mL solution. This activity is the lowest concentration to agglutinate a 2% suspension of human erythrocytes after 1 hr incubation at 25 °C.