Sample Preparation: Buffers

1
Sample Preparation Buffers Techniques

• Bio 3391 L
• Spring 2005
• Ashim

2
Considerations

• Nature of the isolate
• Intact organelles
• Nucleic acids
• Proteins
• Reason for isolation What is it going to be
  used for?
• Source
• If isolating cells, whats the tissue type? E.g.
  get rid of connective tissue if muscle cells are
  needed.
• If isolating organelles- which and for what?
• If isolating proteins or nucleic acids, what
  degree of purity is desired?
• Contamination Possible sources means of
  detection
• Storage conditions

3
Protein extraction
Tissue homogenization
Tissue type animal or plant?

Cytosolic?
Centrifugation
Oragenellar?
Protein source
Normal centrifuge
Differential
Isopycnic
Ultracentrifuge

Protein assay or chemical assays for
determining purity
Collection of fraction
4
Chemical tests Qualitative
5
Assays Quantitative tests

• Nucleic acids
• DNA (ds) Measure absorbance _at_ 260nm (OD of
  150ug/ml)
• RNA Measure OD _at_ 260nm
• Proteins
• Spectrophotometric
• 280nm
• 205nm
• Bradfords assay
• Lowrys assay

6
Buffers

• Buffer A substance that resists a change in pH
  on addition of brief amounts of acid or alkali
• Most are weak acids their conjugate bases or
  weak bases their conjugate acids
• pKa The negative log of the acid ionization
  constant (pKa) is defined as the ability of an
  ionizable group of an organic compound to donate
  a proton (H) in an aqueous media.  The computed
  quantity is a measure of its apparent pKa, or
  macroscopic dissociation constant, at
  equilibrium, normally taken at 25C
• Buffer capacity The amount of an acid or base
  that can be added to a volume of a buffer
  solution before its pH changes significantly.
  (pKa /- 1)

7
Common buffers

• http//www.science.smith.edu/departments/Biochem/B
  iochem_353/Common_Buffers.html

8
Factors affecting buffer action

• Temperature
• Concentration

9
Simple pH titration curves
http//www.chemguide.co.uk/physical/acidbaseeqia/p
hcurves.html