Friday, May 25, 2012

Week 1-Protein Assays and an Assortment of Duties

As the end of my first week approaches, I can already tell that research will be one of the best moments of my undergraduate experience. The environment, my professor, and my research team are all phenomenal and I am so thrilled to be a part of this.

On the first day of lab, I was oriented to the progress in the overall research project that is currently going on.
My Lab Setting

My research activities included making dilutions from more concentrated solutions (or stock solutions) and washing collected tissues with a PBS (saline) solution. I had to be very careful in this procedure because I know my graduate student mentor worked tediously to isolate and collect such delicate tissue. I also participated in making more of a PBS solution and making a dilute solution of the buffer, TBST. 
Washing Collected Tissue with PBS Solution

I also performed a protein concentration measurement with a BCA protein assay kit. Protein assays are performed to measure the total level of protein in a solution. A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknowns before the concentration of each unknown is determined based on the standard curve. The first step in this process is to prepare our diluted albumin standards, known as the BSA. These will standardize our results during the spectrophotometer readings. This entire process is quite lengthy and it requires extremely careful use of several different types of pipettes to insert a precise amount of solute (as low as 2 microliters). The second step in this process is to prepare the BCA Working Reagent which will be used to indicate the concentration of a particular protein in our series of dilutions. The Reagent changes color (green or purple) depending on the concentration of the protein present in the solution. The Reagent is then added to the protein solutions. The third step involves incubating the protein solutions at 37 degrees Celsius for 30 minutes. After incubation, I used a spectrophotometer to measure the absorbance of all the samples. This procedure had to be done very quickly (within 10 minutes) due to temperature-sensitive color development. I finally recorded all the data from my spectrophotometer on an Excel spreadsheet that included averaged values and the actual concentrations (the spectrophotometer reading multiplied by 25 because we performed a 25x dilution to prepare the solution at the beginning). The BSA standards that I prepared are stored in the freezer for later use. Overall, this protein assay matched and confirmed the data values that had previously been collected.

Results Printed from the Spectrophotometer
Protein Samples Stored at 4°C
Eppendorf tubes with Standards and Samples

Protein Assay Kit

The Working Reagent

On the fourth day, my primary focus was transferring the membranes of previously completed Western 
blots into sealed plastic bags. This involved taking the membrane out of its container, transferring it to a plastic bag, adding 0.1% TBST (a buffer) to the plastic bag, and then sealing it with a machine. I cleaned out the black fluorescence boxes that previously contained the Western blots and shelved them so that they can be used for the Western blots that need to be completed in the near future.

Transferred Membranes from Western Blots

On the last day of my first week, I assisted in finishing some of the Western blots. I emptied out the solution in the black boxes that had already been incubated for a certain time, incubated the Western blots with a stripping buffer for 8 minutes two times, then proceeded to wash the membranes with a TBST solution two times for 10 minutes each. For each step, I placed the membranes on a rocker in order to keep the membrane saturated throughout the incubation period. After this, I added a milk solution (1.5 g of Nonfat Dry Milk with 30 mL of TBST) and incubated the membranes in this for an hour. Once the hour was completed, I saturated the membranes in a milk solution+antibodies for 2 hours. After the 2 hours was completed, I washed the Western blots with the TBST solution 3 times for 5 minutes each. Once washing was complete, I incubated the membranes with another milk solution+antibodies for 1 hour.

"Rocker" Machine used to Keep Western Blots Saturated 
Milk+Antibodies used in Western Blotting


That's it for now! See you at the end of next week!

~Lauren Tolat


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