Week 3: Western Blot Analysis

November 21, 2011

Purpose

The objective of this part of the experiment was to transfer the proteins of each plant existing on the SDS-PAGE gel onto nitrocellulose paper so that protein banding patterns can be formed an organized in order to determine quantitatively how much phytochrome A and phytochrome B proteins are present in each of the four different types of plants under blue light.  A western blot is a form of potein analysis in which  

Introduction

 The phenotypic results of the plants under blue light have already been observed, therefore, to understand the full effect blue light has on these four different types of plants, it is necessary to determine how blue light effects the expression of phytochromes A and B, which mediate important plant mechanisms that utilize light to grow and survive.  In a western blot, primary antibodies that bind to phytochromes A and be were added, respectively, as well as secondary antibodies that bind to these primary antibodies and produce a physical banding result that can be see an used to quantify the amounts of hte phytochromes in the plant samples.

Methods

1. 6 pieces of whatman 3mm paper were cut along with 1 piece of nitrocellulose paper.  All were cut to the exact same size as the SDS-PAGE gel.
2. One corner of the nitrocellulose paper was marker with a soft lead pencil so the sides and order of the samples superimposed on the nitrocellulose paper would be known.
3. The nitrocellulose paper was floated on transfer buffer and then submerged.
4. The whatman paper was soaked in transfer buffer afterwards.
5. The gel was rinsed with deionized water.
6. A gel sandwich was constructed in the following order for cell transfer: Positive terminal (+), Western Apparatus Grid, 3 Sheets of Filter paper, Nitrocellulose paper (protein blot), SDS-PAGE gel, 3 sheets of filter paper, Western Apparatus grid, and the negative terminal (-).
7. The transfer unit was run (to red) at the required mA for one hour to complete the transfer.
8. After transfer was complete, the transfer unit was dissembled and the nitrocellulose paper was placed in 50mL of Blotto overnight.
9. The next day, the nitrocellulose was washed with 20mL of TBS-T 1 time for 15 minutes and then 2 times for 5 minutes.
10. The nitrocellulose paper was then incubated in primary antibody solution (TBS-T) until monday, November 28, 2011. 

Conclusion

No results were observed for this part of the experiment since the nitrocellulose paper had to be incubated in the primary antibody solution for a week.  Results will be collected on November 28, 2011.  The anticipated results will show how much phytochrome A and phytochrome B proteins are present within each of the four different plant samples.