Week 2-Solutions and Dilutions

With the completion of my second week, I feel that I am slowly getting adjusted to the lifestyle and work involved in performing research. Many of the graduate students, as well as my fellow mentor, invest long hours into their individual experiments (which are all quite lengthy and cumbersome) in order to meet deadlines. Even though I was warned about the unfortunate "boring" and "slow" part of research, I am amazed at the progress I have been able to witness in the last two weeks. I am also extremely honored to be given the opportunity to work hand-in-hand with such knowledgeable and driven people during my undergraduate experience. I am, indeed, the youngest person to walk around in the lab, but I feel as if I am happily accepted into one big research family.

For the second week, I continued to wash the Western Blots with the TBST solution and saturate them in a milk+antibodies solution. Afterwards, I participated in making an ECL solution. ECL stands for enhanced chemiluminescence and it allows detection of minute quantities of a biomolecule. This occurs because the reaction formed by the mixture emits light when it decays to a singlet carbonyl. Afterwards, we took the Western Blots to a dark room and developed the protein bands on a film. The protein bands are in correspondence to the antibodies that they are saturated with (i.e.-GAPDH).

Substrate used to make ECL

Another set of substrates used to make ECL

Substrate Bottles

Developed Western Blot

I also prepared more TBST solution. This requires a 10x dilution (100 mL of TBST, 900 mL of double distilled water, and 1 mL of a detergent based liquid called Tween). I also prepared more ECL solution and more of a milk+antibodies solution. I also washed the Western Blots (after it had been taken to the dark room to be processed) with TBST two times (5 minutes each) and then a stripping buffer (8 mL for the large box and 5 mL for the two smaller boxes) for 12 minutes. Afterwards, I washed the boxes with a TBST solution two times for 5 minutes each. Last, I added milk+antibodies for 1 hour.

Making TBST-diluting 10x

Making TBST-adding 900 mL of ddH₂O

Making TBST-adding 1 mL of Tween

Making TBST-Final product with an updated label

Making the Milk Solution  

Making TBST-Stirring all contents with this device

Stripping Buffer used in Western Blots