Exam: Lab Content (Notes from Assignments) Flashcards
What is the name of the cell line used in Lab 1? How do these cells grow in a culture flask? What lab space is required to work with these cells? How would you describe their ability to proliferate?
- Ptk2 cells
- adherent
- CL1
- very efficient
What was the experimental question in Lab 1? How did you stress/destress the cells? Why is the unstressed cell sample a negative control?
- Q: How do Ptk2 cells respond to oxidative stress? How does the cell shape change?
- To stress cells, I used 15 microliters (μL) of hydrogen peroxide (H2O2)
- For unstressed cells, 30 μL of distilled water was added (H2O)
- It is a baseline to compare with treatment cells
How does the viability assay distinguish between living and dead cells?
- Living: appear clear or grey due to intact cell membranes
- Dead: stained with blue dye, represented with compromised cell structures
Describe how well you were bale to see the cell shape and colour when you switched the microscope setting between brightfield (O) and phase contrast (Ph)?
- Brightfield: it is hard to see cells/their shape
- Phase Contrast: Ph1 able to see more cells and their structure due to high contrast between dye and actual cells
How do you calculate the concentration of cells? What formula do you use?
cells per mL = # cells / # squares x dilution factor (stock/dilutation x stock) x 10^4
Calculate the concentration of living cells in a tube. There are 28 live cells within 4 hemocytometer grid. You removed 20μL of cells and mixed it with 20μL Trypan Blue.
28 cells / 4 grids x (20μL + 20μL / 20μL) x 10^4
= 7 x 2 x 10^4
= 1.4 x 10^5 cells/mL
Determine how to dilute those cells before seeding them into new flasks:
The ideal cell concentration for seeding should be 1x10 cells/ml . Based on your calculations above, (you calculated 1.4 x 10^5 cells/mL), your live cell concentration is too high! Ack! Therefore, you must dilute your cells before putting them into new flasks
You are starting with 12 ml of cells in media in the collection tube.
Use the dilution formula (C1V2 = C2V2) to determine how much media you will need to add to your tube of cells to create a final concentration of 1x10^5 cells/ml?
C1 = 1.4 x 10^5 cells/mL
V1 = 12 mL
C2 = 1x10^5 cells/mL
V2 = ??
V2 = (C1 x V1) / C2
V2 = (1.4 x 10^5 cells/mL x 12 mL) / 1x10^5 cells/mL
V2 = 16.8 mL
16.8 mL - 12 mL = 4.8 mL
You will need to add 4.8 mL of media.
You prepare your samples and begin to load your gel. Oh no! You notice that the protein sample is not sinking into the well! Instead, it floats in the running buffer. What could have gone wrong with your sample buffer? Explain.
Not enough glycerol was added to the sample buffer since glycerol adds density to proteins allowing them to sink instead of float.
Hemoglobin is a tetramer composed of four polypeptides; two a-chains (each ~16 kDa) and two B-chains (each ~16 kDa). The four subunits are stabilized entirely by non-covalent bonds (i.e. no disulfide bonds).
What is the molecular weight of tetrameric hemoglobin?
4 polypeptides x 16 kDA length = 64 kDA
The molecular weight of tetrameric hemoglobin is 64 kDA.
Imagine that you would like to use gel electrophoresis to observe the size of hemoglobin in its quaternary structure. Suggest three procedural things that you would need to do (or not do) to see the size of the full tetramer.
- Avoid boiling the sample
- Boiling the sample could denature the protein and disrupt the non-covalent bonds holding the tetramer together. - Use a non-denaturing gel like Native-PAGE instead of SDS-PAGE.
- A non-denaturing gel, like Native-PAGE, will preserve the quaternary structure of the protein during electrophoresis. - Use a suitable molecular weight marker
- The marker should have a range that includes the molecular weight of the tetrameric hemoglobin (~64 kDa) for accurate comparison.
If the inhibitory peptide on pepsinogen was only ~100 Da (i.e. 0.1 kDa) in size, and you used only your naked eye, would you see a difference between the inactive and active form on a gel?Why or why not?
- You would NOT be able to see the difference.
- The difference between the inactive and active form is only 0.1 kDA so we could guess its location but it’s so small that it likely wouldn’t present itself.
What is a Ponceau stain? What does it do? Why is it important?
- stains the proteins on the nitrocellulose membrane temporarily
- allows visualization of both control and treatment proteins
- allows us to confirm that cell lysate proteins successfully transferred to nitrocellulose membrane
What is a milk incubation? What does it do? Why is it important?
- coats the nitrocellulose membrane with casein protein
- helps reduce the non-specific binding of antibodies to the membrane
What are the TBS-Tween Washes? What does it do? Why is it important?
- washes the nitrocellulose membrane removing (non-specific) excess antibodies
- this is important to do so that when luminescence rxns are present the sp. spots are dark not the entire membrane
What is Luminol/Oxidizing Reagent Incubation? What does it do? Why is it important?
- it is the substrate for HRP to bind to
- cataylzes a rxn that releases light that we can capture on film, intensity allows us to know about the protein expression while location of the light tells us about the nuclear weight of the protein.
