DRAFT: This module has unpublished changes.

Week 2: Isolation of Protein for SDS-PAGE Analysis and Compilation of Phenotypic Data

November 14, 2011

Purpose

  

The purpose of the experiment this week was to extract an isolate protein from the leaves of each of the 8 plants and perform an SDS-PAGE analysis to determine the quantities of phytochrome A and phytochrome B present in each plant in reaction to the blue light source.  Additionally, the purpose was to compile the phenotypic data measured over week one.

Backrgound

  

Leaves are the part of the plant which absorbs light and has photoreceptors that are used to catch the light source.  Therefore, the leaves of each of the plants are used to obtain the protein source to see how much Phytochrome A and Phytochrome B is present within each plant under the blue light source.  Phytochrome A regulates responses that relate to hypocotyl growth inhibition and cotyledon unfolding that require continuous far red light for maximum expression.  Far red light is also found to induce seed germination (Casal, 2006).  Phytochrome B is thought to play a role in mediation of stomatal opening based on red light regulation (Wang, 2009).  However, blue light is thought to influence the quantity of phytochromes so determination of the amounts of these two proteins can provide insight as to how blue light affects plant growth and phytochrome protein expression overall.

 

Methods

Protein Isolation

  1. 8 tubes were prepared and labeled respectively with the name of each plant.
  2. Two leaves from each of the 8 plants were added to each of the 8 tubes and kept on ice.
  3. In the fume hood, a pestle was prepared by flaming the end of a pipette tip, the end was sealed by gently smashing it into a microfuge tube.  A pestle was prepared for each of the 8 samples.
  4. Working on ice, the tissue of the plant leaves of each plant were ground directly in the appropriately labled microfuge tubs
  5. 1mL of QB buffer was added to each sample and the tissue was ground further on ice.
  6. Samples were spun at top speed in the microfuge at four degrees Celsius for 15 minutes.
  7. The liquid supernatant was transfered into a new tube for each plant sample.
  8. Spinning occurred for a second time to prevent any excess tissue from being included in the isolated protein samples.

Concentration Determination of Extracted Protein

  1. 24 microcentrifuge tubes were obtained so that three dilutions of each of the 8 plants could be labeled as follows: 1:2, 1:20, 1:200.
  2. Each of the extracted protein sample dilutions was prepared in a 100microliter final volume with the addition of QB buffer.  Then for each plant species, the following three dilutions were made: 1:2, 1:20 and 1:200.

Assay Procedure

  1. A 1mg/mL stock solution of Bovine Serum Albumin in QB was obtained.
  2. 6 microcentrifuge tubes were obtained and labeled 1-6.
  3. The standards were prepared by adding the appropriate amounts of buffer, protein standard and unknown protein samples as shown by Table 1. in the procedure handout.
  4. Once the samples were made, they were vortexed immediately.
  5. The required amount of protein determination reagent was prepared by adding 1 part (100microliters) of Copper II Sulfate Pentahydrate (4%) into 50 parts (10mL) of Bicinochoninic Acid solution.  Contents were mixed thoroughly.
  6. A clear 96-well plate was used to load 10 microliters of standards and unknown protein samples into the appropriate wells.
  7. 200microliters of protein determination reagent were added to each well.
  8. The plate was incubated for 30 minutes at 37 degrees Celsius.
  9. The Molecular Devices UV Max Kinetic microplate reader was used to determine the concentration of the uknown protein from the preparation, the wavelength used was 560nm.
  10. A standard curve was prepared by plotting the average protein standard concentrations vs. the net absorbance values at 560nm.  A line of best fit was drawn.
  11. Through use of excel, a linear regression was performed and the slope, intercept and R squared values were recorded.
  12. The concentration of the unknown samples were determined based on the plot and linear regression.

 

Electrophoresis

  1. An SDS-PAGE gel was prepared.
  2. Through use of QB buffer, the quantified samples were diluted out to the same concentration as the most concentrated sample.
  3. The samples were denatured at 95degrees for 10minutes.
  4. 30microliters of the diluted protein sample wasd mixed with 15microliters of the loading buffer. 
  5. As much sample as possible was loaded onto the gel for each protein sample.
  6. The gel was run at 100v for one hour.  The gel was then placed in a washing tray with a few milliliters of gel runner buffer to keep the gel moist.

 

Calculations

The equations C1V1=C2V2 was used to determine which dilution factor yielded the best protein concentration for usage.  The 1:20 dilution factor was determined to be the best dilution factor to use.      

 

Results

The following results are those collected from the phenotypic observations of week one. 

  

  

 

 

 

 

 

 Conclusion

The protein samples will be used for a western blot analysis in week 3, so no results or conclusions exist yet for that part of the experiment.  The Phenotypic results from week one however, show that the non-purple plant had the highest average percent grwoth and the lowest average leaf area.  The pruple plant had the lowest overall percent growth and the largest average leaf area.  These results support the hypothesis concerning blue ligth and show that the pigmented plants absorb less blue light than those without pigments, such as the non-purple plant.

 

 

 

 

 

DRAFT: This module has unpublished changes.