Research Update: Exercise and PGC1a Expression

The first few weeks of my research have consisted of practicing a lot of the techniques that will be used to conduct this research. Since I will be using Western Blotting to determine whether PGC1a’s expression is greater in exercised rat aortas, it is important that I can first make sure that I can run a successful Western Blot of PGC1a with other control tissues from start to finish.

I started off by homogenizing a few different control tissues: mouse aorta, lung, hind limb, and heart. After conducting a few unsuccessful western blots of the aorta and lung (with PGC1a, gapdh, and Cx37), it was determined that perhaps something went amiss in the protein homogenization step. After further review, we realized that the Lysis buffer used to homogenize the protein was over 5 months old! This is not good considering that the Lysis Buffer components only keep for about a month (and working Lysis solution is good for about a day). Having discovered this, I have made the new buffer and homogenized 2 new mouse aorta with this for test controls.

Following the homogenization of the protein, I measured the concentration using a BSA dilution (5ug/ul, 2.5, 1.25, 0.625, and 0.3125) for comparison. It turns out that the first time I did this, the BSA solution, though kept in the freezer, was too old, and caused the protein measurements to be skewed. I made fresh BSA for the next time. I was also measuring the concentration by simply looking at the darkness of the sample and estimating using the BSA dilution for reference.

Colormetric Assay:

Screen Shot 2016-05-18 at 2.23.38 PM

After homogenizing the 2 mouse aortas, I measured their protein concentration using the microplate reader in the lab next door, instead of just estimating by eye. This provided a much more precise measurement of the protein in the two aorta, since it creates a standard curve of the known concentrations of the BSA dilutions in order to extrapolate the protein concentration of the unknowns (the aorta). For more information on what a standard curve is, see the following video on YouTube https://www.youtube.com/watch?v=0luczWOo0rQ. Through this program, we can see that the correlation coefficient of the standard curve is 0.97, which is very good.

Screen Shot 2016-05-19 at 3.00.15 PM

In addition to the standard curve, the program is able to generate a variety of different reports including raw data, absorbance of the samples, and unknown concentrations.

Before I go on to testing my real exercised tissues, there are a few things I’ve learned so far that will be crucial to my success:

  1. Always make sure the reagents you’re using are fresh: Put a date on EVERYTHING. If you aren’t sure how old something is, it’s safer to just make it new, so you don’t waste hours (or days) on something that isn’t going to work.
  2. Pay close attention to the ponceau stain after the transfer: If it doesn’t transfer well then it might not even be worth it to incubate it with the primary antibody (especially since some of the one’s I’m using are rather expensive). Below is an image of a decent transfer—we can see distinct bands with good spread. There are a few air bubbles, which isn’t ideal, but they are not around the target bands I was looking at.

transfer

  1. Manage time well and plan ahead: Also, always leave some time at the end of they day to clean up the lab. I’ve started making sticky notes of all the things I would like to get done in lab during that week. It helps me stay on track and plan out which days I have to do which experiments, since steps like incubating the membrane in the primary antibody have to be done overnight.

I’m currently working on getting the PGC1a antibody to be expressed properly in my sample practice tissues. I’ve still been running into some problems so I’ve been thinking about changing things like antibody concentration and wash time in the imaging protocol. If you have experience with Western Blotting/LiCor imaging let me know if you have any advice or tips for me! Once I can get a solid expression of PGC1a in these tissues, I will be ready to move forward!

-Kayla

Comments

  1. cdong03 says:

    Hi Kayla,

    Congrats on working out the techniques! It’s always super frustrating when an experiment fails because of errors that could’ve been avoided. I’m actually pretty interested in your experiment set up: how are the amount of exercise measured, and what is considered “exercise” for mice? Also I’m wonderig what exactly is PGC1a, and what are its functions? Is it expressed only in certain tissues or globaly?
    Good luck on your research!!

    Chen

  2. Hi Chen,

    Thank you for your comment! My research advisor is actually the one who did all of the exercise training and tissue collection previously. Interestingly, based on the tissues she had, we came up with the idea for this project. The control rats were placed in a cage with a locked running wheel, while exercised rats had access to a running wheel with a counter that tracked the distance they ran each day for 48 days. This is probably the easiest way researchers have developed for exercise training mice and rats, though other methods do exist and are common. I discussed PGC1a in my abstract a little– PGC1a is a gene involved in vascular health and is thought to be involved in mitochondrial biogenesis and stress resistance. There are certain tissues that it is more greatly expressed in (this is a current topic that researchers are exploring), but we know it to be expressed particularly in aorta and adipose tissue!

    Kayla