Western Blot: Technique, Theory, and Trouble Shooting

Western Blot: Technique, Theory, and Trouble Shooting

 

Abstract

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Keywords: 
Bio-medical research, protein, western blot
 

Introduction

Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.

The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present. The paper will first describe the protocol for western blot, accompanied by pictures to help the reader and theory to rationalize the protocol. This will be followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems.

Technique

Cell lysis to extract protein

Protein can be extracted from different kind of samples, such as tissue or cells. Below is the protocol to extract proteins from adherent cells.

Adherent cells:

 

  1. Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).

  2. Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.

  3. Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.

  4. Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).

  5. Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.

  6. Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.

  7. Measure the concentration of protein using a spectrophotometer.

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    determine the volume of protein extract to ensure 50 μg in each well.

  2. Add 5 μL sample buffer to the sample, and make the volume in each lane equalized using double distilled H2O (dd H2O). Mix well. (Tip: Total volume of 15 μL per lane is suggested).

  3. Heat the samples with dry plate for 5 minutes at 100°C.

 

 

Gel preparation

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