Western Blotting Technique Laboratory Analysis

Abstract

This experiment followed an exact protocol from EDVOTEK® Kit #17. This kit was designed to use western blot analysis to detect proteins from Non-fat powdered milk. The experiment had to be run two times before the expected outcome was reached. However, after the second attempt, it was successful. The gel ran well, and the proteins from the gel were successfully transferred

to the membrane provided in the kit.

Introduction

The name western blot was given to the technique by W. Neal Burnette and is a pun on the name Southern blot. Southern blot was a name given by Edwin Southern to a technique he developed for DNA detection.

Western blot analysis has the ability to detect one specific protein in a mixture of any number of proteins while at the same time giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. However, this method does require the use of a high-quality antibody directed against a desired protein. During western blotting procedures, generally, proteins are solubilized with detergents and reducing agents, and then are separated in polyacrylamide gels. After the separation, the pattern of proteins from the gel is then transferred to a nitrocellulose filter or polyvinylidene fluoride membrane. The proteins then become covalently bound to the support. Western blots and Southern are distinguished from one another by the probe. In western blots, specific unlabeled antibodies, which are typically specific to each individual protein and will therefore react specifically with the target protein. This is what allows for the recognition of the protein of interest from within the background of other cellular proteins. The bound antibody is then detected on the blot by a secondary reagent. This reagent can be either radiolabeled or coupled to an enzyme. Western blots can be used to search for the presence of certain proteins in specific samples, tissues, or treatments. They may also be used for quantitative analyses or to determine the apparent molecular weight of a protein in a sample.

Experimental Procedures

This experiment follows the protocol provided with EDVOTEK Kit #17. All procedures are taken directly from that protocol, with no deviations.

Preparation of Reagents (letters refer to labeling in Kit #317)-

all references to water indicate that biochemically pure water was used.

Transfer Buffer Solution

● add 100 mL of 10X Tris-glycine concentrate to 700mL of H2O

● add 200 mL of 95-100% MeOH to the above solution- stored tightly sealed.

Electrophoresis Buffer

● make a 1-10dilution of the EDVOTEK 10X buffer with H2O

Reconstitution of Lyophilized Proteins

● 125mL pure water added to each of the protein tubes, wait 30sec for the proteins to fully dissolve

● suspend protein tubes in a boiling water bath for 10 minutes,

Immunodetection Reagens

● Add 540mL water to all of the provided the 10X blocking buffer

● Add powdered milk (J) to 600mL blocking buffer (I)

● dilute anti-BSA antibody 1:1000