DRAFT: This module has unpublished changes.

 

Week IV: Development of Western Blot for Final Data Analysis

Tuesday, November 22nd, 2011

Purpose

The purpose of this week's experiment is to develop the Western Blots in order to determine the amount of phytochrome A and B present in the Brassica rapa after being subjuected to high intensity LED red light (and five other light regimens) for one week's time.  

 

Introduction

The SDS-PAGE gels that were subjected to a transfer treatment last week will be subjected to a series of washes, a primary antibody treatment, and a fluorescently-tagged secondary antibody treatment. Upon analysis of the fluorescence which will signify the presence of phytochrome A and B, an analytical comparison will be able to be made of the amount of phytochromes present between each of the Western Blots produced by other groups whose Brassica rapa plants were subjected to different LED light regimens during week one.

Procedure

  • Wash nitrocellulose paper with 20 mL of TBT-T once for fifteen minutes and twice for five minutes each.
  • Incubate nitrocellulose paper in an antibody solution of 10 microliters in 20 mL of TBS-T for 90 minutes. 
  • After the 90 minutes, wash the nitrocellulose membrane with 20 mL of TBS-T for 30 minutes and then twice more for 15 minutes.
  • Within the last few minutes of the second wash, mix a preparation of 750 microliters of both CD1 and CD2 and then slowly add it to the nitrocellulose paper until bands appear.
  • Once the appearance of bands is presented on the gel, rinse the nitrocellulose paper with deionized water.

Results

Unfortunately, the analysis of the WEstern Blots was not possible because the images produced did not present any bands.  Because bands were not observed, the presence of phytochrome A and B was not able to be quantified from the study.

What we had expected to see for our group's light regimen in particular was a greater amount of phytochrome B produced, since phytochrome A, which absorbs red light, is converted to phytochrome B in the plant.

One plausible explanation for the human error presented from our results is that accidental human tampering of the plants may have occurred when collecting data. By measuring the stem length, some of the plants were uprooted because the plants were rather small and difficult to measure with a ruler. The uprooting of the plants would result in the plants’ limited ability to obtain water and nutrients from the soil, thus hindering the growth of the plants.  Also, the measurements taken may have had some inconsistencies in that different group members took the measurements each day.  Each group member may have had a different methodology of rounding the measurements taken because the marks on the ruler were rather difficult to distinguish.

Overall, the presented hypothesis was rejected based on human error that occurred while collecting data for this study. If we were to be more cautious on measuring stem length and surface areas of leaves, substantial increase in the Brassica rapa plant growth over the week may have occurred. Another aspect of the experiment that could improve the efficiency of the results produced would be a more clear interpretation of the results obtained from the Western Blot. If more consistent measurements within solutions along with the placement of the nitrocellulose papers were done accurately, we could possibly depict the phytochrome A and B levels within our Brassica rapa plants exposed to high intensity red light. 

DRAFT: This module has unpublished changes.