Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Appendix I: Glossary
Absorbance – The amount of ultraviolet light that is not transmitted (absorbed) through a sample. 280 nm
is the ultraviolet wavelength absorbed by the tryptophan, tyrosine, and phenylalanine groups in a protein in
solution. The relationship between absorbance of the ultraviolet light and protein concentration is linear.
Affinity chromatography – A chromatography method of separating molecules based on a highly specific
biologic interaction such as that between an antigen and antibody, enzyme and substrate, or receptor and
ligand.
Anion – A negatively charged ion or biomolecule.
Anode – Positive electrode; attracts negative ions.
Anion exchange chromatography – A chromatography method where a positively charged
chromatography resin binds negatively charged molecules, or anions.
Aromatic amino acid groups – Amino acid groups that contain an aromatic ring in the R-group
are nonpolar and absorb untraviolet light at 280 nm. Aromatic amino acids are tyrosine (Y, Tyr),
tryptophan (W, Trp), and Phenylalanine (F, Phe).
β-mercaptoethanol – (BME) is a reducing agent used to break the diusulfide bonds of proteins, thus
disrupting the tertiary and quaternary structure of the protein. It helps to linearize the protein in prepartion
for electrophoresis.
Bradford Protein Assay – A test used to measure protein concentration in a sample. The assay relies on
the shift in absorbance of Coomassie Brilliant Blue G-250 dye. The dye reacts with mainly basic amino acid
and aromatic amino acid groups.
Buffer – The liquid that is used to dissolve the biomolecules that will be applied to the chromatography
column.
Cathode – Negative electrode; attracts positive ions.
Cation – A positively charged ion or biomolecule.
Cation exchange chromatography – A chromatography method where negatively charged
chromatography resin binds positively charged molecules, or cations.
Cell lysate – All the components, soluble and insoluble, of a cell that have been broken open.
Centrifugation – Spinning a mixture at very high speed to separate heavy and light particles. In protein
expression and purification, centrifugation results in a “pellet” found at the bottom of the tube, and a liquid
“supernatant” that resides above the pellet.
Charge density – The protein’s ratio of charge to mass.
Chromatogram – A visual output of the chromatographic separation. Peaks on the chromatogram
indicate when samples are eluting from the column.
Appendix I: Glossary
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APPENDIX I
GLOSSARY