SDS/PAGE

Question 1

What is the separation in SDS-PAGE based on?

Answer 1

Movement of charged proteins in an electrical field and sieving effects

Question 2

What is SDS/PAGE an analytical tool for?

Answer 2

• Determining the purity of protein preparations
• Approximate molecular weight determination
• Western blot analysis

Question 3

What is the migration of proteins approximately proportional to?

Answer 3

Their mass

Question 4

Which molecules will have greater mobility with same charge density?

Answer 4

Small

Question 5

How can proteins be visualized?

Answer 5

1) Staining (e.g., Coomassie or silver ions) - all proteins
2) Chemiluminescence - Antibody-based detection - specific target protein

Question 6

What are the titles for the x- and y-axis when determining the concentration of an unknown protein?

Answer 6

x - relative migration
y - logMr

Question 7

What are the objectives of this lab?

Answer 7

• Efficient casting of SDS-PAGE gels
• Proper sample application and gel running
• Visualization of proteins by Coomassie staining
• Analysis of successful E. coli expression of PTEN (IPTG induction success; cell lysis efficiency; solubility analysis)

Question 8

What does proteomics aim to identify?

Answer 8

The protein complement of a particular cellular state

Question 9

What is proteomics useful for?

Answer 9

• Deciphering organelle-specific protein
• Mapping functional networks

Question 10

What was a biomarker discovery of proteomics?

Answer 10

Differential expression analysis

Question 11

How can gel electrophoresis be employed within proteome studies?

Answer 11

As a separation step to reduce sample complexity and visualize potential biomarkers

Question 12

How is protein separated in 2D gel electrophoresis?

Answer 12

Weight and isoelectric point

Question 13

What does 2D gel electrophoresis combine to give?

Answer 13

Combines isoelectric focusing and SDS-PAGE to give a fingerprint of protein expression in a given cell

Question 14

What happens in the first dimension of 2D gel electrophoresis?

Answer 14

Isoelectric focusing gel with decreasing pI is placed on SDS polyacrylamide gel

Question 15

What happens in the second dimension of 2D gel electrophoresis?

Answer 15

SDS polyacrylamide gel electrophoresis with decreasing Mr from top to bottom and decreasing pI from left to right

Question 16

What is DIGE?

Answer 16

Difference gel electrophoresis - quantifying technique for protein purification

Question 17

How is DIGE carried out to overcome non-biological variation?

Answer 17

1) Label protein extracts using CyDye DIEGE Fluor minimal dyes
2) Mix labelled extracts
3) 2D separation
4) Image gel with CyDye specific scanner (scan at different wavelengths)
5) Image analysis and data quantitation using appropriate software

Question 18

What are the advantages of fluorescent detection?

Answer 18

• Increased dynamic range
• Spot co detection
• Accurate (corresponding spots have the same boundary, ensuring exact spot volume rations)

Question 19

What is the proteomics 2D gel-based workflow?

Answer 19

1) Sample prep
2) Sample loading
3) Protein separation
4) Image acquisition
5) Image analysis
6) Automated spot picking
7) Spot digestion
8) MALDI target spotting
9) Spot handling workstation

Question 20

In a scientific report, what did chronic stepwise cerebral hypoperfusion do?

Answer 20

Differentially induced synaptic proteome changes in the frontal cortex, occipital cortex, and hippocampus in rats