Lectin Staining Thoery

Lectin Staining Theory:

Lectins are proteins derived from plants, animals, or microorganisms that bind to specific terminal sugar residues in carbohydrates. The binding is affected by the structure of the carbohydrate chain so that even small changes within the carbohydrate molecule can cause changes in the binding of lectins. Lectins with the same terminal sugar specificities, therefore, do not necessarily show the same binding patterns. Lectin histo-chemistry can thus detect small changes in the composition of glycoproteins which can also be used in the study of normal cellular differentiation and also in the study of malignant cells.

Lectin Staining Material/Methods:

Using the protocol for lectin staining provided for this lab session, the methods described were followed. First, the prepared blot was rinsed twice 3 minutes apart with lectin buffer (LB), after which, it was blocked in LB/1% BSA for one hour at room temperature. The blot was then washed 3 times with LB spacing the washes out by 3 minutes. Biotinylated lectin solution was then added and incubated for an hour, followed by another washing (3x3 minutes). Blot was again washed (3x3) with PBS containing 0.1% Tween 20, and then incubated for another hour in ABC reagent, and again washed with PBS as before. The color was then developed with DAB reagent and required the measurements taken for the identification of the glycoprotein.

Immunoblot theory:

Western blot or immunoblot is an analytical technique utilized for the detection of specific proteins in a given tissue homogenate or extract sample. Gel electrophoresis is a part of the technique in an attempt to separate native or denatured proteins via the size of the polypeptide chains or three-dimensional configuration of the proteins. In this particular case SDS-PAGE is used. The data collected on the gel is then moved or transferred to membrane where they detected using antibodies that are specific to the protein in question. The polypeptides are transferred for this particular lab electrophoretically to the membrane (nitrocellulose or PVDF) for detection.

Immunoblot Protocol:

The immunoblot or western blot protocol is as follows:

The blot was rinsed with PBS-0.1% Tween 20.

Then blocked