Module 3 ALL of the methods for proteins, DNA, and RNA

Salting out

Advantage: simple, cheap, quick first-pass separation. Disadvantage: low resolution, separates crudely by solubility. Used: early/crude step to concentrate protein fractions.

Dialysis

Advantage: gently removes small molecules (like salt) without harming the protein. Disadvantage: doesn't separate proteins from each other. Used: removing salt/buffer after salting out, before further purification.

Gel-filtration chromatography

Advantage: separates by size, gentle, preserves native structure. Disadvantage: limited resolution for similarly-sized proteins, dilutes sample. Used: separating proteins by size or estimating native mass.

Ion-exchange chromatography

Advantage: high resolving power based on charge, reusable columns. Disadvantage: requires knowing the protein's charge properties, salt gradients can denature sensitive proteins. Used: separating proteins by net charge (cation or anion exchange).

Affinity chromatography

Advantage: extremely specific, high purity in one step. Disadvantage: requires a known ligand for the target protein, can be costly to develop. Used: purifying a specific protein via a tag or natural ligand.

HPLC (high-performance liquid chromatography)

Advantage: very fine beads give high resolution and fast, sharp separations. Disadvantage: requires high-pressure equipment, more expensive. Used: when maximum resolution and speed are needed.

SDS-PAGE

Advantage: simple, fast, accurate mass estimate, visual via staining. Disadvantage: denatures the protein, no info on native structure/activity. Used: determining protein mass and checking purification purity.

Isoelectric focusing (IEF)

Advantage: high-resolution separation by isoelectric point (pI). Disadvantage: only separates by charge, not size. Used: determining a protein's pI.

Two-dimensional electrophoresis

Advantage: combines size (SDS-PAGE) and charge (IEF) for very high resolution. Disadvantage: technically demanding and time-consuming. Used: comparing whole-proteome expression patterns under different conditions.

Ultracentrifugation

Advantage: determines mass, density, shape, and interactions of native (non-denatured) proteins. Disadvantage: requires specialized, expensive equipment. Used: studying native protein properties via sedimentation coefficients (S values).

Polyclonal antibodies

Advantage: easier and cheaper to produce, recognize multiple epitopes. Disadvantage: batch-to-batch variability, less specific. Used: general detection when high specificity isn't essential.

Monoclonal antibodies

Advantage: highly specific, consistent, unlimited supply from immortal hybridoma cells. Disadvantage: more costly and technically complex to generate. Used: when a highly specific, reproducible antibody is required.

ELISA (enzyme-linked immunosorbent assay)

Advantage: quantifies protein amount with high sensitivity via colorimetric readout. Disadvantage: requires a specific antibody, possible cross-reactivity. Used: quantifying protein or antigen concentration in a sample.

Western blotting

Advantage: combines electrophoresis and antibody detection to identify a specific protein by size. Disadvantage: semi-quantitative at best, multi-step and time-consuming. Used: detecting or confirming a specific protein after electrophoresis.

Co-immunoprecipitation

Advantage: identifies protein-protein binding partners under native conditions. Disadvantage: only detects fairly stable interactions, can pull down non-specific binders. Used: identifying binding partners of a protein of interest.

X-ray crystallography

Advantage: atomic-level resolution, well-established and widely used. Disadvantage: requires a well-ordered crystal, cannot detect hydrogen positions, gives only a static single conformation. Used: when atomic-detail structure is needed and a good crystal can be obtained.

NMR spectroscopy

Advantage: studies proteins in solution in their native, dynamic state, can capture multiple conformations. Disadvantage: limited to relatively small proteins, requires high protein concentration. Used: studying structure and dynamics of smaller proteins without needing a crystal.

Cryo-electron microscopy (cryo-EM)

Advantage: no crystal needed, works for large complexes, captures multiple orientations at once. Disadvantage: historically lower resolution than X-ray (though improving), computationally intensive reconstruction. Used: determining structures of large proteins or macromolecular complexes that are hard to crystallize.