Lab 8

Question 1

What does post-translation modification cause?

Answer 1

Phosphorylation
- Alter activity, localization and degradation
- Phosphorylation sites can be predicted
- Reduces isolecletric point

Question 1

SDS-PAGE depends on what?

Answer 1

Molecular weight, charge, shape

Question 2

What is important in SDSPAGE (what does SDS do>)

Answer 2

Sodium doecyl sulfate
- Removes structure ( boiling)
- SDS unfolds protein
- SDS coats it to remove charge-MW ratio

Question 3

Stacking vs separating gel

Answer 3

• Stacking gel has less acrymalide
• Stacking - protein is sandwiched between Cl and glycine
• In separating, glycine is in the front with Cl

Question 4

3 sections to lab

Answer 4

Run protein gel
Analysis
Transfer (WB)

Question 5

2 types of SDS-PAGE

Answer 5

• 1D (separation on MW)
• 2D (separation on charge, then MW)

Question 6

What is mini-PROTEAN apparatus?

Answer 6

Gel electrophoresis tank for protein separation
- Vertical, with cathod at top and anode at the bottom

Use for SDS PAGE

Question 7

Semi-dry apparatus

Answer 7

Transfer and immobilize proteins from an SDS-PAFE onto a membrane (PVDF)
Membrane will detetc specific POI

Question 8

What does SDS do?

Answer 8

To separate proteins based on MW, SDS
1) Denatures proteins
2) Coats protein to create a net negative charge proportional to MW

Question 9

What is stain-free technology for band visualization?

Answer 9

• Exposure to UV using ChemiDoc
• Activates trihalo, which covalently modifies tryptophane in protein - causes fluorescence

Only modified proteins are visible as the trihalo compound by itself does not produce any fluorescence

Question 10

What are the two types of gels in SDS-PAGE?

Answer 10

• Stacking gel (ensures all proteins enter gel at same time)
• Resolving gel (separates proteins by molecular weight)

Question 11

Why doesn’t trihalo create background fluorescence?

Answer 11

Isn’t fluorescent unless bound to proteins

Question 12

Two types of Western Blot

Answer 12

• Wet: gel and membrane fully submerged, for large, slow moving proteins
• Semi-dry: run faster, sandwiched between gel and membrane

Question 13

How does Western Blot transfer work?

Answer 13

Voltage is applied to transfer apparatus so that proteins (coatede in SDS) move toward positive membrane

Question 14

What is in a PAGE?

Answer 14

• buffer
• gly- and cl-
• anode, cathode
• polyacrylamide matrix

Question 15

What are the two chambers in a PAGE?

Answer 15

Vertical chamber where upper buffer is cathode and lower chamber is anode