Merck, L6397-5MG, Concanavalin A from Canavalia ensiformis (Jack bean)

Application
Detects glycoproteins containing α-D-mannose, α-D-glucose when used with appropriate peroxidase substrate
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)

Biochem/physiol Actions
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.
Packaging
Package size based on protein content
Physical form
Lyophilized powder containing tris-citrate buffer salts and trace calcium and manganese
Preparation Note
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.
Analysis Note
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.
Other Notes
One unit will form 1 mg purpurogallin in 20 sec from pyrogallol at pH 6.0 at 20 °C.