Advance Cell Bio Lab FINAL
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What is used in buffer for PAGE?
DDT TRIS (6.8) Bromophenol Blue SDS Glycerol
What DTT do?
Breaks disulfide bridges externally and internally
What Tris (6.8) do?
To keep the pH of the sample 6.8
What brophenol blue do?
Tracking dye for SDS-Page
What SDS do?
To denature the proteins by breaking down the cell wall and giving the protein a negative charge
Glycerol
To add density to the protein samples
What proteins were we studying?
Muscle proteins, actin
How do different layers of page function?
Stacking layer has a pH of 6.8 making glycine a zwitter ion and SLOW (allowing proteins to be stacked) Running layer has a pH of 8.8 making glycine negative and FAST
What was used to extract the proteins?
Laemmli sample buffer
What type of membrane was used for western blotting
Nitrocellulose membrane
What role do HCI play in SDS-PAGE?
They move quickly down in the stacking layer and remove any ions attached to the proteins
How does electric work in transferring?
The electricity allows the proteins to be blotted onto the membrane in a mirror image
How does western blot work?
Primary antibodies attach to the antigens than the secondary anti bodies attach (this is crosslinked with HSP) a insoluble substrate (hydrogen peroxide) is needed (unlike ELISA)
What is the percent of the q30?
It shows the quality of the sequence based on an average 1 per 1000bp
What is the percent of pf?
Measure of flow cell don’t crossover and readable (we want low)
Why dont we want overlap? And how do we get it?
When they are overlapped they are not able to be read, this is caused by non diluted DNA
Where do BHK-21 cells come from?
They are derived from baby hamster kidney cells
What are FBS?
Serum of growth hormones from fetal calf that induce survival and proliferation
What is the minimal medium?
MEM (minimum essential medium) this contains minerals, AA, sugars etcs (USE TO SUSTAIN GROWTH)
Why do we not use a CO2 incubator?
We used carbonic acid to balance the pH, this is already found in the medium
What is used to wash and collect? (harvest)
We use trypsin (from goat stomach) or PBS (3 washes). It allows the cells to be suspended. Collects dead cells
What is cell passaging?
The process of transferring growth stage cells from one medium to a fresh growth medium (allowing further propagation and maintenance of the cell line)
What is camptothecin?
An anticancer drug, it binds to the topoisomerase I and DNA complex preventing DNA replication imitating apoptosis
What occurs when apoptosis is started?
Instability within the membrane (phospholipid symmetry is ruined) PS is exposed (leaves!!)