This week, you get to write the lab procedure. What you will be doing is using SDS polyacrylamide gel electrophoresis to look at each of the samples you saved during your reaction center isolation. You will also be performing a Peterson analysis of the total protein concentration of each sample. Here are the points to consider in designing your protocol (a written protocol with amounts and as detailed as possible an account of what you will do is your prelab this week):

1) You have five hours. You had better arrange the experiment so that while the gel is running other things are getting done. You should also arrange it so that you have at least three hours allowed for running the gel.

2) The small gels that we are going to use have a volume of about 10 mls each for the stacking and running gel. There are 10 slots per gel (for loading sample), each about 4 mm wide by 0.75 mm.

3) Think about the chromatophores you ran last time on the gel and their concentration. You will not have time to complete the Peterson assay before running the gels so you will need to use your previous information (what you know about protein concentration vs. absorbance in chromatophore samples) to estimate how much chromatophore and each subsequent sample to load on the gel. Given the absorbance you now see in your R-26 chromatophore samples, what protein concentration would you estimate you are working with? As you go on to the samples from later in the prep, should you load the same volume or a greater volume on the gel? Think this through carefully.

Your prelab for this week is a written procedure for what you will do.

1) Look at the gel results. Try to estimate approximately how many protein bands are visible at each step of the reaction center isolation. How many subunits does the reaction center have (based on your data)? What are their molecular weights?

2) Using your differential spectra at each step in the isolation as a measure of reaction center activity and your Peterson analysis to determine the total amount of protein present, determine the relative specific activity of the reaction center at each step (call the specific activity of the reaction centers in the initial chromatophore sample 1.0 and express the specific activity at each subsequent step relative to that).