Week 3: Western Blot Analysis

Monday, November 21, 2011

Group 3 (Tori, Samantha, Stephanie, Jared, Karafaye)

Purpose

The purpose of this week's experiment is to perform a Western Blot analysis in order to determine which proteins, particularly phytochromes, are present in the Wisconsin Fast Plants after being exposed to high intensity red light for one week.

Introduction

A Western Blot is performed in this week's lab in order to determine how much phytochrome A and phytochrome B is present in the Wisconsin Fast Plants after being exposed to high intensity red light for one week.  Primary antibodies from phytochrome A and B are obtained from the SDS-PAGE gel and they are added to the nitrocellulose paper.  The addition of secondary antibodies to the nitrocellulose paper allows bands to be observed, but this step will occur in week 4.

Procedural Outline

• 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

Proteins from the SDS-PAGE gel from phytochrome A and B were obtained are added to the nitrocellulose paper.  Next week, secondary antibodies will be added to the nitrocellulose paper in order to develop the Western Blots and see the appearance of bands.