Protein Analysis

Question 1

What is pathology of Sickle cell?

Answer 1

Single substitution at pos.6 of Beta dimer (Glutamate to Valine)

Question 2

How many charges does the substitution in a SS pt cause?

Answer 2

Glu->Val change in charge by -1.

But beta unit forms dimer, so net change is -2

Question 3

What is SDS role in SDS-Page Electro..

Answer 3

SDS covers linear chain with neg charge.
Denatures 2’ and non-disulfide 3’ structures

Differs from the reducing agent

Question 4

What is an epitope?

Answer 4

Portion of a molecule to which an Ab binds

Question 5

What indicators are used to spot Ab-Ab bound regions in protein analysis techniques?

Answer 5

1. Alkaline phosphatase

2. Horseradish peroxidase

Question 6

What are ELISA tests used for?

Answer 6

Detection of Abs/antigens in pts:

1. Home pregnancy tests
2. Detection of Ab to virus
3. Biomarkers for MI

Question 7

What is the difference btwn direct and indirect ELISA?

Answer 7

Direct-expensive, indicator is developed based on 1’ Ab

Indirect-less expensive, indicator is based off a secondary Ab (more generalized)

Question 8

How are HIV false positives avoided?

Answer 8

Confirm positive ELISA test with positive western blot.

Question 9

Does SDS break disulfide bonds?

Answer 9

No, we need a reducing agent to do that.

Question 10

In a HIV ELISA what is immobilized on the plate?

Answer 10

HIV antigens. We are looking to see if we have HIV Abs in blood.

Question 11

Why is nitrocellulose (nylon) paper used for blotting?

Answer 11

Typically positively charged. Allows neg proteins/DNA/RNA depending on your blot, bind with paper

Question 12

What are epitopes composed of?

Answer 12

Sugars/Lipids/AAs

Question 13

What are applications of Western Blot in medical Dx?

Answer 13

HIV
Lyme
HBV
HSV
Duchenne muscular dystrophy

Question 14

What is the window period in HIV?

Answer 14

Time between the virus in the blood and being able to detect Abs

Question 15

What do we look for to dx HIV from a Western blot?

Answer 15

No bands - Negative
Bands @p31-OR-p24 AND bands at gp160-OR-gp120 -> (+)
Bands present but not at those seq. -> indeterminite (retest in 6wks)

Question 16

What is a signaling molecule that can be picked up. On ELISA post-MI

Answer 16

Troponins (troponin I most specific)

Question 17

What is co-immunoprecipitation? What is a case where we use this method?

Answer 17

Being able to discern two molecules that may inherently be linked. Commonly used for p53 typing. I.e p53 is a tumor supressor, but when MDM2 (an E3 ubiquitin ligase) is upregulated this binds p53 and puts it out for degradation. This leads to less p53 and more tumor.

We can immunoprecipitate mdm-2 with anti-mdm2 serum and we may very well see p53 proteins that coprecipitated.

Question 18

What is immunoaffinity chromatography?

Answer 18

Purifying proteins from cell extracts (affinity purification), then we can isolate and identify interacting proteins.
We have a column and line it with Abs. Add sample of interest, then run what sample you have left thru SDS-Page

Question 19

What is immunohistochemistry used for?

Answer 19

Mostly for looking at samples from pt under a slide. Similiar process to ELISA in that we use ABs to target specific proteins in tissue. And a secondary Ab as an indicator. But we are just looking at these under a slide.

Clinical EXAMPLE:
HER-2 (HUman Epidermal GF Receptor 2) in 1/5 breast cancers. Tend to be more aggressive than other breast cancers. More HER2 will be stained than in a normal tissue cell.

Question 20

What is an epitope tag?

Answer 20

Usually A.A.s

Added to molecule to help visualize (in a gel/western blot/immunofluorescense)