Week 6: Clone Screening - Alexandra Kot

 Alexandra Kot | Dr. Sandra Demaria 

2026.07.06:

As my cells were not confluent enough, I waited until Monday to irradiate them. Both of the knocked-out proteins, MAVS and MLKL, are induced by radiotherapy under normal conditions. Therefore, to analyze whether a knockout occurred, the cells need to be irradiated so the resulting protein levels are above the level of detection. Each of my 78 clones received 8Gy of radiation over three minutes. To allow for an adequate response, we will wait 48 hours before performing the cell lysis steps. 

2026.07.07:

This morning Dr. Jason Spector, Chief of the Division of Plastic and Reconstructive Surgery, spoke to us on bioengineering at the interface of research and medicine. Though it seemed like Dr. Spector's talk was going to go deeper into the engineering advances being used/discovered in the field of plastic surgery, most of the talk focused on the history of plastic surgery and some of the more interesting cases he had been involved in. He outlined the reconstructive ladder, consisting of healing by secondary intention, primary closure, skin graft, flaps, and free tissue transfer. It is truly incredible the kind of reconstructive capabilities we have and how resilient the human body is. 

I prepped four gels to be used for tomorrow's Western blot. 

2026.07.08:

Now that it was 48 hours after irradiating the cells, I spent five hours of my day doing lysis on each of my knockouts. The process involved washing off media, adding radioimmunoprecipitation assay (RIPA) buffer, which disrupts the cell membranes to allow for protein extraction, using a cell scraper to remove adherent cells, centrifuging, and then transferring the supernatant. Though the plan was to run 48 Western samples today, the lysis steps took so long, so it makes more sense to start the Western blot tomorrow. 

Figure 1: Most of my samples on ice, with RIPA. Samples needed to be kept on ice to prevent protein degradation. 

2026.07.09:

Today was for setting up and running my four western blots a total of 28 samples from each knockout line. I denatured samples with laemmli buffer and loaded samples into each slot. Set the machine to 70V to allow the samples to align in the stacking buffer and then to 100V to run through the separating buffer. I staggered the ladder in each of the different gels to ensure that I could identify each set of samples. After which, I performed a wet transfer, stained with ponceau, and blocked the plots with a primary antibody to sit overnight. 

2026.07.10:

After adding the secondary antibody and imaging, I was able to obtain these western blots as shown below in Figure 2. It appears as though the guide we have to knockout MAVS is relatively poor. However, we expect a potential knockout in samples 6, 7, 26, 27, 28. I will screen these clones again against a control clone (#1, which still expresses MAVS really well) next week. On the other hand, the MLKL knockouts went really well with knockouts in samples 3, 5, 15, 24, 13, and 14. I will expand #13 and #14 today and will freeze the remaining knockouts and place them in storage. 

Figure 2: MAVS and MLKL knockout western blots


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