Week III: Western Blot Analysis

Monday, November 21st, 2011 

Purpose

The purpose of this week's experiment is to perform a Western Blot in order to determine which proteins, in this case, phytochromes A and B, are present in the Brassica rapa plants after being exposed to high intensity LED red light (and five other light regimens) for one week's time.

Introduction

A Western Blot is performed in this week's lab in order to determine how much phytochrome A and phytochrome B protein is present in the Brassica rapa after being exposed to high intensity red light (and the five other light regimens for the class data) for one week's time.  The SDS-PADE gel that was run last week was obtained out of safe storage.. The analysis of the Western Blot will be conducted during week four.

 Procedure

• Cut six pieces of whatman paper and one piece of nitrocellulose paper in the exact size of the SDS gel.
• With a pencil, make a mark on the corner of the nitrocellulose paper.
• Place the gel, the whatman paper, and the nitrocellulose paper in a glass container and submerge in transfer buffer.
• Rinse the gel with deionized water.
• A sandwich was created with the materials in the following order:

POSITIVE TERMINAL (+)

WESTERN APPERACTUS GRID

3 SHEETS OF FLITER PAPER

NITROCELLULOSE PAPER (PROTEIN BLOT)

SDS-PAGE GEL

3 SHEETS OF FILTER PAPER

WESTERN APPARATUS GRID

NEGATIVE TERMINAL (-)

• Run the transfer unit at the required mA for one hour to complete the transfer.
• Once the transfer has completed, place the nitrocellulose in 50 mL of Blotto overnight in preparation for next week's lab.

Conclusion

At the end of this laboratory period, SDS-PAGE gels had finished experiencing the transfer and were then subjected to an overnight Blotto soak. The gels were stored appropriately in anticipation of an antibody treatment to be conducted during week 4.