Nov 23, 2020
Cytochrome C Assay
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. Cytochrome C Assay. protocols.io https://protocols.io/view/cytochrome-c-assay-bpz6mp9e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 23, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44830
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Steps
Steps
Start ON cultures of a strain of interest.
The next day, measure 100 mL BHI into each flask to be used.
Measure OD600 of each overnight and calculate how much of your overnight culture you need for OD600~ 0.01in 100mL.
Note
TKO – 20 min – WT – 10 min – DKO
Add supplement if doing Long Term exposure or place BHI + 0.01 cells directly into 37*C for a later spike-in
Harvest cells at 0.3 if doing Long Term or 0.225-0.25 if doing Spike
Note
Check OD after 2.5 hours.
Obtain OD600 values for each of your cultures and then calculate how much of your culture is needed to reach an OD600 of 7 in 3 mL
Note
- Formula: (7-OD600)(3mL) = (measured log phase-OD600)(X mL)
- Will need around 60-80mL to achieve this concentration
Pipet half the total volume needed into a 50mL conical and centrifuge for~10 minutes at 3500 RPM. Pour off supernatant. Add the other half of the cell volume.Centrifuge again.
Wash the cells with 10 mL of 20mM MOPS buffer*(stock is 1M) by centrifugation for 5 min at 3500 RPM. 20mM MOPS buffer is made in H2O
Note
C1V1=C2V2 formula to figure out how much stock to make 20mM.
*MOPS-3-(N-Morpholino)propane sulfonic acid resuspend in H2O– autoclave.
Repeat the wash
Resuspend the pellet in 3 mL of 20mM MOPS buffer
Add 1mg/mL of Cytochrome C (stock is 10mg/mL in H2O)- vortex
Note
C1V1 = C2V2
Incubate for 10 minutes
Centrifuge for 10 minutes at 3500 RPM
Add 2 mL of the supernatant to 4mL plastic cuvettes
While cytochrome cells are spinning, make standards. To 3 mL of 20mM MOPS buffer add:
- 0.1 = 30uL of cytochrome
- 0.2 = 60uL of cytochrome
- 0.3 = 90uL
- 0.4 = 120uL
Use the Cytochrome program (“CYTOCHROME”) on the Fozo Lab Spec = OD530
Note
Standards listed above cover a range of predetermined mg/mL results, if your results fall outside of this range, you may need to adjust your standards accordingly.