Analysis Note

Agglutination activity is expressed in µg/ml and is determined from serial dilutions in phosphate buffered saline, pH 6.8, of a 1 mg/ml solution. This activity is the lowest concentration to agglutinate a 2% suspension of human blood group O erythrocytes after 1 hour incubation at 25 °C.

Application

General Western Blot Protocol: Glycoprotein sample size: 500ng Lectin Concentration: 0.1ug/mlLoad 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

Ulex europaeus agglutinin has anti-H blood group specificity. There are two types of lectin: UEA I has an affinity for L-fucose and UEA II has an affinity for N,N′-diacetylchitobiose. The mol. wt. of UEA I was initially found to be 170,000 daltons. Later reports indicate that UEA I may form aggregates at neutral and basic pH and that the correct mol. wt. is 68,000 daltons.

Linkage

Conjugates are prepared from affinity purified lectin.

Packaging

0.5, 1 mg in serum bottle

Package size based on protein content.

Physical form

Contains sodium citrate buffer salts

Preparation Note

Prepared from peroxidase, Type VI (P8375), using the two-step glutaraldehyde method of Avrameas and Ternyck.Repurified by affinity chromatography after conjugation.

Unit Definition

One unit will form 1.0 mg purpurogallin in 20 sec from pyrogallol at pH 6.0 at 20 °C.