Lab Flashcards
(28 cards)
Why is a standard curve required to measure the unknown protein concentrations
because it serves as a reference for measuring the unknowns
How do we ensure equal loading of protein in each gel lane?
- we used a spectrophotometer to calculate the absorbance of the samples and compared it to standard curve.
- That allowed us to calculate the diluted then undiluted concentrations, using that number we calculated the volume of each sample needed to equal 50ug of protein
Why was it important to add SDS to our samples and gel?
- SDS denatures, unfolds, and covers protein in a negative coat.
- This allows protein to move more accurately according to it’s molecular weight in gel electrophoresis
During loading, if any sample spilled over to neighboring lanes, how might this affect your interpretation of the results?
if any sample spills over it could lead the neighboring lane to migrate more or less than it should
what does the coomassie stain do?
binds to all proteins and stains them blue
What is predicted molecular weight of FAM171B
92kD
How did we move our proteins from gel to nitrocellulose membrane
- electrophoretically
- nitrocellulose is cut to gel size and a “transfer sandwich is set up”
Directly after the transfer, how might we be able to tell if our membranes appropriately received protein?
If we can see the ladder on nitrocellulose membrane the protein should have transferred as well
Should we be able to “see” FAM171B at this point (after immunoblot)
- No, we can’t tell where FAM is based on Coomassie gel.
- We need something more specific since it stains all proteins we don’t know what one is FAM (so we do antibody staining)
what is the purpose of blocking reagent
it increases how specific our antibody staining is by covering/ block empty spaces on the membrane
what is the primary Ab that we are using and what is it’s purpose
- We are using R FAM171B as a primary antibody.
- It’s function is to cross the membrane and bind to FAM171B. This allows the secondary antibody to bind to the primary antibody
How did we block and add primary Ab
- the nitrocellulose membrane was washed with distilled water in a meal bag, the water was then squeezed out of the bag
- after 3mL of TBS-T block buffer was placed within the meal bad an the air bubbles pushed out
- following this the bag was sealed with the nitrocellulose membrane and buffer
- after incubating for 1hr the secondary antibody is added and then re-sealed
why must sterile conditions be maintained in a cell culture lab?
to ensure culture contamination doesn’t occur
what are cells fed as they grow in vitro?
EMEM media with 10% FBS which has all the nutrients the cell needs to grow
how do we detach the U138 cells from the T25 flask?
a PBS/EDTA solution is used which inhibits some membrane-associated proteins leading the U138 cells to detach from the flask
Why grow cell at 37 C with 5% CO2
the cells have evolved to live in that environment and if taken out of those conditions pH of the media will no longer be neutral and cells will become alkaline
what will these cells on coverslips be used for
immunofluorescence experiments
what was the purpose of washing the membrane prior to secondary Ab?
the purpose of washing the membrane prior is so that antibodies inappropriately bound to proteins are washed off
what does the second Ab do?
it binds to the primary Ab and since the second Ab has a dye it allows it to be seen with immunofluoresnces
Is FAM171B a nuclear or cytoplasmic protein?
it’s a cytoplasmic protein
What further test could be performed to confirm your results after a Western blot?
immunofluorescence
what was the purpose of the MeOH step in immunofluorescence
- stabilize and fix cellular protein to wherever they’re located
- punctures the cell’s membrane to let the Ab through
why block the coverslip?
to increase specificity of the Ab staining of the protein
what is happening during the primary antibody step?
the antibody diffuses around the cell and attaches to FAM171B
