Developing a citrate synthase assay protocol

Time to do something I haven’t done in a while: learn a new lab protocol from scratch! Mikaela, Clara, and I are working on developing a citrate synthase assay protocol for the Abcam colormetric kit. Here’s what’s been done so far:

• Found my old Github issue asking Olivia about the assay
  • In this issue, Olivia provided her protocol and some helpful hints
• Jasmine modified Olivia’s protocol for clarity and put together a Bradford Assay protocol based on a tutorial from Pascale
• Mikaela and Clara created a kinetic measurement protocol on the plate reader based on the modified protocol

2026-07-27

Today our goals were to:

1. Create protein lysates
2. Conduct a Bradford Assay

When grabbing the reagents from the fridge, I noticed a discrepancy in our protocol. The kit reagents very clearly need to be stored at 4ºC, but the modified protocol said that reagents should be stored in the -20ºC. Additionally, there were different reagents listed in the protocol vs. what we received! I did some digging for the actual kit protocol from Abcam. We followed the directions from the manufacturer’s protocol with some changes that Mikaela, Clara, and I will work on changing in the protocol itself:

Protein lysate preparation:

1. Remove flash-frozen tissue from the -80ºC and place immediately on ice
2. Cut and measure 10-20 mg of tissue per sample and place in a new tube
3. Add 350 µL of the Extraction Buffer into the tube with the issue. Homogenize the tissue using a pestle
4. Incubate homogenates on ice for 20 minutes.
5. Centrifuge homogenates for 20 minutes at 4ºC and 16,000 rcf
6. Transfer the supernatant into a new tube. Place in the -80ºC for 6 months or measure immediately in a Bradford assay

Notes:

• Each sample has roughly ~20-30 mg of tissue, so we aimed for 10 mg of tissue for this run to have something to work with in case we needed to redo anything.
• HOMOGENIZING TISSUE CONTINUES TO BE MY LEAST FAVORITE THING IN THE WORLD. Olivia’s protocol said to use a p1000 to homogenize, so I figured that the sample could be pipet homogenized or mashed effectively with a pipet tip. This was…..not the case. I ended up getting some reusable glass pestles which worked a lot better
• The glass pestles displaced a lot of volume in the 1.5 mL tubes, so we transferred the tissue and buffer to a 5 mL tube to have more room for homogenization. Next time, we will only add 100 µL of buffer for homogenization, then top off with 250 µL of buffer to avoid using too many tubes.
• It took us 20-30 minutes between sectioning tissue to making homogenates. Samples were on ice or in the Extraction Buffer at all times, but I’m worried about how much protein is left in those samples.

We didn’t get to the Bradford Assay today, but we will do it with Pascale tomorrow.

2026-07-28

Today we did the Bradford Assay with Pascale. Jasmine made our existing protocol based of a tutorial with Pascale earlier this summer, but based on today’s tutorial they are modifying the protocol to improve clarity and reproducibility. Here are a couple of things we learned:

• The Bradford Assay reagent is very sensitive to temperature! Yesterday Pascale took the reagent out for us since it needs to be brought to room temperature prior to doing the assay. Since we didn’t end up using it, I put it back in the fridge and took it out today before we ran the assay. Our protocol said it needed to come to temperature for 20 minutes prior to using it, but we learned that we don’t want to take it out until we’re actually loading the plate. Otherwise, it’ll “go bad,” which is indicated by it no longer being a brown/swamp monster color.
• If we add the assay reagent to the wells with the standards and we don’t see a nice temperature gradient across the samples, then something is wrong with either the assay reagent or our standards and they need to be redone. If our standards don’t look good, then we don’t add the reagent to our samples!
• We quantified protein based on an undiluted sample and a 1:2 (lysate:total volume) dilution.
• We used the Extraction Buffer as our assay blank. Seems like there’s something in the Extraction Buffer that is being quantified.

Based on the quantification, we got 0.4-0.9 mg/mL. I think we can improve our concentrations by 1) using 175 µL of Extraction Buffer instead of 350 µL and 2) having a more solid lysate production procedure! Mikaela and Clara are going to repeat the Bradford Assay with four new samples: two from the 16 ppt treatment, and two from the 5 ppt treatment. This way, we can finalize our lysate and quantification protocol and they can proceed to quantify the rest of the samples.

Going forward

1. Quantify protein content with Bradford assay
2. Finalize protein lysate-making methods
3. Try controls and samples in full assay protocol
4. Troubleshoot protocol as needed