BCMB 320 - Midterm

Explain the concept and principle of vapour diffusion

Protein sample and reagent (precipitant) is placed in vapour equilibration with reagent. Water leaves drop - ends up in reservoir. sample undergoes supersaturation (protein increase in concentration). Protein starts to precipitate very slowly allowing the protein molecules to align themselves in a consistent orientation.

What is circular dichroism

A spectroscopic technique used to analyze the secondary structure of proteins by measuring the differential absorption of left and right circularly polarized light.

explain the principles of circular dichroism

It involves the differential absorption of circularly polarized light by chiral molecules, providing information about protein secondary structures such as alpha-helices and beta-sheets.

Explain the principles of Cryo-Electron Micrscopy

Sample of the structure of interest is flash frozen in vitreous ice and kept frozen while being observed with the electron microscope. Useful to determine the molecular structure of large, dynamic, macromolecular complexes and integral membrane proteins.

Explain the principles of NMR

Carried out on molecules in solution. captures dynamics of protein structure: conformation changes, protein folding, interactions with other molecules. Measures nuclear spin angular momentum, which generates a magnetic dipole which alines in 1 of 2 orientations: parallel (low energy), or Antiparallel (high energy). Pulse of electromagnetic energy generates an NMR Spectrum.

Explain the principles of Mass spec.

provides information on molecular mass of proteins, amino acid sequence of proteins, and information on entire proteomes. Separates analyses according to their mass-to-charge ratio (m/z).

Steps of mass spec (4)

1. Ionize analytes in a vacuum.

2. introduce charged molecules to electric and/or magnetic field.

3. charged molecules move through field as a function of the mass to charge ratio.

4. deduce mass of analyte.

Describe the ionization methods for protein mass spectrometry

Different techniques used to convert proteins into ions, such as Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). These methods facilitate the analysis of proteins by generating charged particles that can be measured by mass spectrometry.

describe the structure and function of the magnetic sector

• longest MS in use

• Voltage accelerate ions into a magnetic sector

• ions deflected in magnetic field

• ions detected base on m/z ratio

  • lighter ions with same charge experience more deflection.

Describe the structure and function of time of flight MS

• accelerator move ions by applying a voltage between two electrodes.

• Ions with a smaller m/z ratio hit detector earlier

describe the structure and function of Ion trap MS/Orbitrap

• ions are trapped in an electric field where they oscillate

• ions with unstable oscillations are discharged and hit detector.

Describe the structure and function of Quadrupole MS

• Ions with stable oscillations travel through to detector

• Unstable ions collide with electrodes and discarded.

what are the basic principles of purifying lipids

Use organic solvents as they disrupt hydrophobic interactions between lipids.Lipids can be separated based on their solubility in different solvents, allowing for selective extraction and purification. For neutral lipids, use non-polar solvents (diethyl ether, or chloroform). For polar lipids, use polar solvents (ethanol, and methanol).

use solvents that are peroxide free to ensure no oxidation of lipids

• Organic solvents react with oxygen (air exposure) to produce peroxides.

what are the principles of absorption chromatography

lipids in mixtures can be separated based on their polarity and interact with polar materials such as silica, using absorption chromatography methods such as HPLC or TLC.

Solid material (absorbent) attracts solutes through van Der Waals forces and H-bonds.

describe the principles of TLC

Separate molecules between the mobile and stationary phase.

Stationary phase: silica immobilized on aluminum/plastic plate.

Mobile phase (Eluent/ solvent): usually a mixture of polar and non-polar solvents.

Separation depends on affinity of analyte for mobile phase.

The higher the affinity of the analyte for the mobile phase the further the analyte is carried on the TLC plate.

Explain Heterologous gene expression

Expression of a gene from one species in a different species.

How is a crude extract prepared when purifying a protein.

1. Lyse cells via osmotic lysis (cells are suspended in a high salt buffer, water diffuses out of the cell, then it is transferred to pure water, and lyse because of the sudden influx of water, homogenization (rotor stair homogenizer, fast and efficient but not gentle), grinding (handheld glass tube and piston, smaller volumes), sonication (sonicator, sound waves break membranes and weakened cell walls previously treated by enzymes, intensity can be adjusted to ensure lysis but not denaturation of proteins).

2. Centrifuge to separate soluble proteins from cell debris.

what are the key considerations when preparing a crude extract

want to stabilize proteins using:

1. Detergents: Ionic and non-ionic detergents

2. maintain cooler temperatures to reduce denaturation.

3. use appropriate buffers for optimal pH for the protein, and don’t interfere with protein activity.

4. ionic strength: protein solubility either maintained or salted out.

5. metal ions: many proteins require metal ions as cofactors. some heavy metals can negatively impact protein activity.

6. oxidation: Free -SH groups susceptible to oxidation, want to prevent this.

7. proteases: Proteases degrade proteins, prevent by including protease inhibitors.

explain centrifugation and the different types of rotors and centrifuges.

Centrifugation is a technique used to separate components in a mixture based on density by spinning the sample at high speed. Different types of rotors include fixed-angle, swinging-bucket, and vertical rotors, each suited for specific applications, while centrifuges can vary in capacity and speed.

Most widely used centrifuges: Microcentrifiuge (most without refrigeration, 1.5ml and 2ml tubes), bench top (refrigerated, variety of rotors, 10ml to 500ml tubes/bottles), Ultracentrifuge (refrigerated, variety of rotors, mostly 40ml Tubes)

Explain the theory of chromatography

Chromatography is a method for separating components of a mixture based on their different interactions with a stationary phase and a mobile phase.

explain different types of elution strategies and mobile phase changes

Isocratic - no change in counter ion concentration

Gradient - gradual increase in counter ion concentration

Step-wise - sudden increase in counter ion concentration

explain the principles of Gelfiltration/size exclusion chromatography (which molecule elutes first, what can the gels be made of?)

• separates molecules by size, smaller molecules move into stationary phase and are slowed down - take longer to elute

• larger molecules elute first

• collection different size molecules in different fractions.

• Most resin/gels are made from agarose, dextran, or polyacrylamide.

explain the principles of Ion exchange chromatography (which molecule elutes first, what can the gels be made of?)

• Cation or anion exchange chromatography

• separates based on charge

• Cation exchange chromatography

  • stationary phase has negative charge

  • binds positively charged molecules

  • negatively charged molecules elute first.

• Resins/gels are derivatives dextran or agarose.

  • DEAE (diethylaminoethyl) anion exchange resin.

  • CM (Carboxymethyl) cation exchange resin.

explain the principles of affinity chromatography

• Based on specific interactions between a protein and a ligand.

• Ligand is immobilized on the stationary phase

• specific protein binds to ligand on stationary phase and is retained on the column.

• proteins not bound to ligand elute first

• Elute protein by increasing free ligand concentration either gradient or step-wise.

explain the concepts behind improving resolution

Ability to separate depends on relative rates of migration.

Two approaches

• Increase band separation (separate peaks)

• Decrease band width (Narrow peaks)

Can manipulate several chemical and physical parameters to achieve this, such as flow rate, temperature, and column dimensions.

Explain the principles of the Bradford assay

• Uses coomassie brilliant blue G-250 dye.

• G-250 binds protein through hydrophobic and ionic interactions

• abs max change from 465nm to 595nm when bound to protein

• observed as a visible colour change (brown/red to blue)

• Requires an acidic environment to maintain the anionic form of the dye.

Explain the principles of the BCA assay? what does BCA stand for?

• BCA - Bicinchoninic acid assay

• In alkaline environment proteins reduce Cu2+ to Cu 1+

• Subsequent binding of 2 bicinchonic acid molecules with Cu 1+ produces an intense purple colour.

explain how proteins are quantified with the Bradford and BCA assays

Both use a standard curve to determine the protein concentration.

Bradford assay is linear between 0-30ug protein, while BCA assay is linear between 20-2000ug/mL).

Explain the advantages and disadvantages of the Bradford assay

Advantages:

• easy, fast, and sensitive

• can be used for a mixture of proteins

  • proteins from cells fraction and samples for electrophoresis.

Disadvantages:

• Protein used for STD curve is different than those in your sample

• Agents bind differently

  • Primary structure of proteins are different

  • binds primarily Arg but also His, Lys, Phe, Tyr, Trp.

Explain the advantages and disadvantages of the BCA assay

Advantages:

• standard curve is linear over a large working range (20-2000 ug/mL)

• Detergent compatible whereas Bradford assay is not

Disadvantages:

• variability between proteins (Trp, Tyr, Cys - colour formation)

• No true end point (colour may still develop slowly after incubation

• take slightly longer than Bradford assay.

Explain how proteins are stored appropriately

• “shelf life” is protein specific

• Degradation of protein due to:

  • Microbial and proteolytic degradation

• Need to use storage conditions that prevent degradation

  • Important to use sterile equipment

• Certain storage conditions can cause protein to lose structural integrity and function (activity).

• best to store proteins in concentrated solutions ( >1mg/mL

• Put additives that will not affect subsequent use of protein samples:

  • Cryoprotectants - 25-30% glycerol and ethylene glycol.

  • Protease inhibitors - proteolytic degradation

  • anti-microbial agents - NaN3

  • Metal chelators - EDTA, prevents metal induced oxidation of - SH groups

  • Reducing agents - DTT (dithiothreitol), 2-mercaptoethanol

What is the shelf life of a solution at 4c? how many times can the sample be used?

Shelf life: 1 month max

Times sample can be used: many

What is the shelf life of a protein in 25-50% glycerol or ethylene glycol at -20c? how many times can the sample be used?

Shelf life: 1 year

Times sample can be used: many

What is the shelf life when frozen at -20c or -80c or stored in liquid N2? how many times can the sample be used?

Shelf life: years

Times sample can be used: once

What is the shelf life when lyophilized? how many times can the sample be used? what does lyophilized mean?

Shelf life: years

Times sample can be used: once

Lyophilized means freeze dried.

Explain the principles of electrophoresis

Electrophoresis = Migration of ions in an electrical field

Explain the principles of SDS-page (what does SDS-page stand for? applications)

SDS = Sodium-dodecyl-sulfate

PAGE = Polyacryamide gel electrophoresis

Applications:

• Separates proteins by size

• Estimate the purity of your protein

• determine protein size

• blotting applications

• protein identification

• rough estimation of protein quantity

Explain the principles of SDS-page (how does it work?)

• SDS coat proteins to produce negatively charged molecules with an even charge density to length ratio (1 SDS molecule/ 2 amino acids)

• B-mercaptoethanol added to reduce di-sulifde bonds ( required to form “rod” shape)

• separate proteins by size using electrical current

• Negatively charged proteins move towards positive electrode.

• ammoniumpersulphate

  • Disulphate ester of H2O2

  • generates SO4- radicals

• SO4- reacts with TEMED (tetramethylethylenadiamine) to induce polymerization of acrylamide

• Acrylamide

  • Water-soluble monomer

  • used to produce 3D-Lattice (gel)

  • Needs cross-linking agent

• Bisacrylamide

  • Cross-linking agent

• Polymerization occur by free radical chain mechanism

• free radical generated by chemical decomposition.

What is Discontinuous gel electrophoresis? what are the names of the different gels? what is the pH of the buffer? what is the charge of the Gly?

• two different gels in acrylamide concentration and different pH

  • Stacking gel and resolving gel

Buffer

• pH 8-9

• Gly mostly neg. charge carry electrical current

What does the stacking gel in discontinuous gel electrophoresis do? what is it made of and what pH?

• Acrylamide concentration is not restrictive

• keeps protein sample concentrated.

• 2-3& acrylamide

• pH 6.9

• Gly twitter ion mostly, low mobility

• proteins and Cl migrate faster, higher mobility

What does the Resolving gel in discontinuous gel electrophoresis do? what is it made of, what concentration of acrylamide, what pH?

• protein separation

• pH 8-9

• Gly shifts back to negative charge

• Gly and Cl carry current

• Acrylamide concentration >10%

Explain the principles of IEF (also what does IEF stand for?)

IEF = Iso Electro Focusing

• Many side chains of amino acids in proteins are charged

  • Depends on the pH

• Therefore, proteins have a charge and an isoelectric point (pI)

• When setting up a pH gradient between an anode and cathode, protein will migrate to the pH corresponding to their pI.

• pH gradient setup by molecules called ampholytes

  • contains amino and COOH groups

  • ionize at different pH

  • Range of pI’s.

• With electrical current, ampholytes migrate to pH =pI, setting up pH gradient

  • more acidic ampholytes near anode, with more basic toward cathode.

• IEF gels often contain 6M urea as denaturing agent

  • NOT SDS, as it gives proteins negative charge.

Explain the principles of 2D gel electrophoresis

Combines IEF with SDS-PAGE.

Protein lysate is put through IEF and the SDS-Page.

Name the different staining methods and their use

• Lava purple stain

  • Fluorescent molecule (epicocconone) that reversibly binds to lysine, arginine, and histidine residues.

  • Works for 1D and 2D gels, and can be used on PVDF and nitrocelluclose membrane (Blotting application).

  • Highly compatible with MS.

  • Environmentally safe.

• SYPRO ruby staining

  • Designed for 2D PAGE

  • good for SDS-Page and IEF

  • Relatively expensive

  • Ultrasensitve, similar to Ag-staining

    • simpler and faster than Ag-staining

    • does not interfere with Edman degradation or Mass spec.

  • COOmassie blue staining

• Silver staining

Explain how coomassie blue staining works

• Coomassie R-250

• Detection limit 0.1-0.5 ug protein.

• binds primarily Arg but also His, Lys, Phe, Tyr, and Trp.

Explain how silver staining works

• is more sensitive than coomassie blue

• detection limit

• 0.2-0.6ng protein

• Ag-staining more laborious, expensive and requires meticulous work

• method

  • fix protein with methanol

  • gels can be stored for several days in fixing solution

  • Treat gel with Na-Thiosulfate

    • sensitize the gel and prevents AgCO3 precipitate formation when AGNO3 is eventually added.

  • silver ions binds proteins

  • silver reduced to metal form by addition of formaldehyde in developing solution (Na2CO3)

    • Do not over develop

    • can stop by adding 5% acetic acid

  • Transfer to 1% acetic acid for long term storage