What are the steps of protein purification starting with tissues or cells?
- Cell lysis
- Protein binding to matrix
- Washing
- Elution
How should total protein and specific activity change as you purify?
Total protein should decrease as activity increases
What are the different column chromatography techniques?
- Size exclusion/gel filtration
- Ion exchange
- Affinity
Size exclusion/gel filtration chromatography
Separates proteins based on size
Ion exchange chromatography
Separates proteins based on net charge
- Uses 2 matrices: DEAE and CMC
- DEAE: positively charged, anion-exchange matrix
- CMC: negatively charged, cation-exchange matrix
Affinity chromatography
Separates proteins based on binding affinity to ligands
SDS PAGE
Way to separate denatured proteins by charge and size (final step to determine purity)
Western blot (immunoblot)
Used to detect proteins separated by gel electrophoresis
- Uses primary (protein-specific) and secondary (detection) antibodies
What do SDS and beta mercaptoethanol do in SDS PAGE?
- SDS PAGE adds a negative charge to all the proteins
- Beta mercaptoethanol breaks disulfide bonds
What is the role of monoclonal antibodies?
- Homogeneous Ig species that recognize 1 epitope on an antigenic protein
What is the role of polyclonal antibodies?
- Heterogeneous mix of Ig proteins that recognize 1 or more epitopes on an antigenic protein
What reserach methods use antibodies?
- Western blot (immunoblot)
- Affinity chromatography
- Immunofluorescence
- Immunoprecipitation
- Co-immunoprecipitation
- ELISA
ELISA (enzyme-linked immunosorbent assay)
- Identifies low level antigenic proteins
- Direct and indirect ELISA
Immunofluorescence
- Used to identify proteins in cells that have been chemically treated in a way that preserves the cell’s architecture
- After washing, cells can be visualized through fluorescence microscopy
What are the advantages of NMR vs X-ray crystallography?
NMR advantages
- Good for proteins still in solution
- No need to crystalize
- Good for studying dynamics of proteins during catalysis
X-ray crystallography advantages
- No theoretical size limitation
Practice question: What is the correct order to to isolate and organelle from a tissue if the organelle isn’t as dense as the nucleus?
- Obtain tissue
- Break open the cells to obtain a homogenate
- Centrifuge at a low speed
- Take the supernatant and centrifuge at a great speed for a longer time to obtain a second pellet
Practice question: What are the purposes of SDS and beta mercaptoethanol in SDS PAGE electrophoresis?
- SDS gives all the proteins a negative charge
- Beta mercaptoethanol breaks disulfide bonds
Practice question: Which technique is the best to access purity of your protein?
SDS PAGE
Practice question: DNA was attached to chromatography resin and then the proteins that bind DNA were isolated. This is an example of ___.
Affinity chromatography
Practice question: As you purify a protein, you want ___ to increase while ___ decreases.
- Activity
- Total protein
