Protein electrophoresis

Question 1

Different types of PAGE (polyacrylamide gel electrophoresis)

Answer 1

• SDS-PAGE
• Blue Native PAGE: characterise large proteins in their native and active forms
• Zymogram PAGE: characterise collagenases and proteases

Question 1

Functions of SDS in SDS-PAGE

Answer 1

SDS = sodium dodecyl sulfate
Imparts:
1. overall negative charge on the proteins
2. similar charge-to-mass ratio for all proteins - binds at consistent rate of 1.4g SDS per 1g protein (1 SDS molecule per 2 amino acids)
3. long, rod-shape on protein instead of complex tertiary formation
Tf, SDS-bound proteins migrate based primarily on their sizes, allowing MW determination

Question 2

Theory of zymogram PAGE

Answer 2

Gels are cast in gelatin or casein, which act as substrates for the collagenases/proteases. After separating the enzymes(proteins) by MW in SDS (denaturing condition), the enzymes are renatured, allowing breakdown of substrate. Staining with Coomasie blue R-250 will leave clear areas around active proteases.
Emphasis on ACTIVE proteases

Question 3

What is Isoelectric Focusing (IEF)

Answer 3

IEF combines an ELECTRIC FIELD with a PH GRADIENT to separate proteins acc to their pI (isoelectric point).
Offers highest resolution of all electrophoresis techniques

Question 4

2 methods of generating a stable, continuous pH gradient for IEF

Answer 4

1. Carrier ampholytes - heterogenous mixtures of small conductive polyamino-polycarboxylate compounds with closely spaced pI values. When voltage applied, they align themselves acc to their pI and buffer pH in their proximity
2. Immobilized pH gradients (IPG) strips - by covalently grafting buffering groups to polyacrylamide gel backbone

Question 5

Types of Gentle cell disruption methods

Answer 5

1. Osmotic lysis (in hypotonic solution - cells swell and burst)
2. Freeze-thaw lysis (in liquid nitrogen)
3. Detergent lysis (solubilizes cell membrane, usually followed by sonication)
4. Enzymatic lysis (digests cell walls, followed by sonication)

Question 6

Types of Harsher cell disruption methods

Answer 6

1. Sonication (cooled on ice to avoid overheating)
2. French press (shear force through small orifice)
3. Grinding (pestle and mortar)
4. Mechanical homogenization (hand-held device, useful for small, solid tissues)
5. Glass-bead homogenization - vortexing with glass beads

Question 7

Basic workflow of protein sample preparation for PAGE

Answer 7

1. cell disruption
2. protein solubilization
3. contaminant removal, desalting, concentration
4. quantitation (determining protein concentration)
5. preparation for PAGE

Question 8

What is the process of protein solubilization

Answer 8

Process of breaking interactions involved in protein aggregation eg disulfide bonds, hydrogen bonds, VdW forces, ionic interactions, hydrophobic interactions.
If these interactions not prevented, proteins can aggregate or precipitate = artifact/sample loss

Question 9

Types of reagents for protein solubilization

Answer 9

• Detergents (eg Triton X-100)
• Reducing Agents (B-ME, DTT)
• Chaotropic agents (urea)

Question 10

Different types of discontinuous buffer systems and respective leading (L)/trailing (T) ions

Answer 10

1. Laemmli (Tris-HCl): L = chloride, T= glycinate
2. Bis-Tris: L = chloride, T = MES/MOPS
3. Tris-Acetate: L = acetate, T = Tricine, suited for large protein SDS-PAGE
4. Tris-Tricine
5. IEF

Question 11

Types of total protein stains

Answer 11

1. Coomassie stains
2. Fluorescent stains
3. Silver stains
4. Negative stains
5. Stain-free technology

Question 12

Relationship between migration rate of protein in SDS-PAGE vs its MW

Answer 12

Migration rate of protein coated with SDS inversely proportional to the logarithm of its MW