1. Run SDS-PAGE with 10% resolving gel, 3% stacking gel. Use 50 ug of protein fr
ID: 218693 • Letter: 1
Question
1. Run SDS-PAGE with 10% resolving gel, 3% stacking gel. Use 50 ug of protein from each subcellular fraction. 80V thru stacker, 180V thru resolving.
Why do we run the gel slower thru the stacker, compared to the lower resolving gel?
2. Transfer to nitrocellulose (NC) paper. Place these items in the box in the following order (bottom to top):
A. One white pad.
B. 1 piece 3M paper.
C. Gel
D. NC paper
E. 1 piece 3M paper
F. One white pad
Why is the order of A-F important?
3. Add 50 mls of destain to the stained blot. Shake for 15 seconds. If the background is not light pink, add more destain. Shake for 15 additional seconds. Rinse with DI water for 1-2 minutes. Remove blot from box and place on flat surface. Importantly- using a Sharpie, place a dot "." by each standard band. Take a picture. Rinse and dry the staining box.
What is the purpose of staining the blot?
What is the relationship between the Coomassie stained gel and the Ponceau stained blot?
Explanation / Answer
The purpose os tacking gel is to allow all the proteinns to enter the running gel at the same time. This is achieved as the protein is stacked between cloride ions and glycerate where cloride run ahead of protein and glycerate run behind the protein stacking the gel in between until it reaches the running gel when glycerate becomes more ionized and leaves the protein behind and move with cloride away from protein. Then the proteins of variable sizes are seperated based on their charge. Running without stacking gel results in smeared proteinn on the gel.
If the voltage is kept high at stacker gel, glycerate will move ahead of the protein and a smear would be obtained.
Feel free to leave a comment down below for any further query. Thank you.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.