Lab Methods: Centrifugation, Spectroscopy & Assays

Centrifugation and Spectroscopy

Larger and denser particles sediment faster. Differential centrifugation pellets nuclei, then mitochondria, then microsomes, then ribosomes as speed rises; an isopycnic gradient bands each particle at its own density. Report force in $\times g$, since RCF grows with rotor radius and RPM squared. UV-Vis reads electronic transitions and conjugation, IR finds functional groups (C=O near 1700 cm$^{-1}$), NMR maps H environments, and mass spectrometry gives molecular weight. Beer's law, $A = \varepsilon l c$, turns absorbance into concentration.

Molecular Assays

Southern blots detect DNA, Northern blots RNA, and Western blots protein. PCR doubles the target each denature-anneal-extend cycle, so copies $= N_0 \times 2^n$; RT-PCR starts from RNA. A sandwich ELISA reads antigen amount as color, quantified against a standard curve.

Key Takeaways

• Svedberg values are not additive: 50S + 30S makes a 70S ribosome.
• Beyond about $A = 1$ the Beer's-law line bends; dilute and remeasure.
• Measure mRNA with RT-qPCR or a Northern blot, protein with a Western or ELISA.