Assignment Task:

Investigation of transporter abundance in a variety of human colon samples

Introduction

Western blotting, also known as immunoblotting, involves the immobilization of proteins onto a nitrocellulose membrane (Towbin et al., 1979, PNAS 76: 4350-4354), followed by detection using specific antibodies. This technique can provide information concerning the tissue expression, molecular weight and/or existence of isoforms for the particular protein being investigated. 

The first stage is to use gel electrophoresis to separate the protein samples to be investigated according to size. This is achieved using the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique. Since the SDS binds to all proteins and makes them equally negatively charged, the rate at which a protein runs through the acrylamide gel is totally dependent upon its size (i.e. the smaller the protein, the further it will travel in a given time). 

The second stage is to transfer the protein from the acrylamide gel onto a nitrocellulose membrane. This was traditionally achieved using “wet” tank transfer and took between 2 and 16 hours. More recently, “semi-dry” transfer equipment can be used that can transfer the protein onto the nitrocellulose membrane in less than 10 minutes.

The third stage is to perform the antibody investigation. This begins with incubating the nitrocellulose membrane in a specific 1o antibody (e.g. to detect NHE3, UT-B, CD147, MCT1, NaKATP or DRA) for ~20 hours. Following washing of the membrane, incubation for one hour is performed in the relevant 2o antibody (e.g. if 1o antibody was produced in a rabbit, use an HRP-labelled anti-rabbit 2o antibody). After washing, detection of the specific protein signal is achieved utilising ECL chemilumiscent reagents and a LAS-4000 imager.

 

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  • Posted on : August 08th, 2018
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