Protein Purification Techniques Flashcards
Setting the proteins free
Break up the cells (lyse them) by different techniques
Sonication, homogenization, osmotic pressure
How does protein concentration vary across cells?
No cell contains all proteins possible coded for by the DNA. Some proteins are similar so small or large differences can separate them
Sonication
Room with sonicator (loud room) put sample in sonicator and its blasted with sound waves and cell membranes lyse
Homogenization
Metal ball bearing, machine that shakes it that breaks up cell membranes
Osmotic pressure
Salt, put in water and the membranes blow up
Procedures of homogenation
Break cells with high frequency sound
Use a mild detergent to make holes in the plasma membrane
Force cells through a small hole using high pressure
Shear cells between a close fitting rotating plunger and thick walls of a glass vessel
What is in the homogenate?
Contains large and small molecules from the cytosol such as enzymes, ribosomes, and metabolites, as well as the membrane bound organelles
Wen done carefully most of the membrane bound organelles are left in tact
Taking the cells for a spin (types of centrifugation)
- Differential Centrifugation
- Rate Zonal Centrifugation
- Equilibrium Density Gradients
Sedimentation rate equation
s = m[1-(do/d)]/f
Differential centrifugation
- Differential centrifugation separates matters according to mass and density.
- Can be used to separate organelles and large protein complexes- good for purifying out big things
Process of differential centrifugation
Homogenate filtered many times and then poured it to leave highest density items in previous tubes
Nuclei/ mitochondria, chloroplasts, lysosomes, peroxisomes/ plasma membrane, large polyribosomes, ER fragments/ ribosomal subunits, small polyribosomes/ soluble portion of cytosol(including proteins)
Rate-zonal centrifugation
Sucrose gradient is used to provide density stability during centrifugation
Particles of a similar density remain in the fraction because of mass and shape
Better resolution of separation
You have substances of known density and you separate cell particles by how they fall in this scale. Separate and study fractions
What happens after fractionation?
- What is actually in each fraction collected during centrifugation?
- Utilize column chromatography
Column Chromatography
• Separate mixture ofproteins based on different properties of the protein
- Size
- Charge
- Binding affinity
Gel filtration
Size exclusion chromatography
“Matrix”= stationary beads in column, small proteins trapped by pores in the beads and the flow more slowly than the large proteins that flow around the beads
Large proteins appear in the earlier fractions
May have to do more than once
Ion exchange chromatography
Cation (negatively charged beads) and anion (positively charged beads) columns
Proteins move through columns at rates determined by their net charge at the pH being used
How do you get the proteins out of the ion column?
Elute charged protein with solution (ex: if negatively charge protein, use salt solution)
The added solution outcompetes for the charge and binds to the beads, causing the proteins to come off
Affinity chromatography
Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest
Results in highly specific purification, covalent bonds
Use a competing molecule, salt, or change in pH to remove the protein from the column
Which fraction has the protein of interest?
Assay to determine presence of protein in the fraction
Protein Purification
• Purification techniques to isolate a specific protein from all other cellular components
Basic steps in protein analysis:
• Protein assays (why start with an assay?)
• Protein purification
• Look for specific activity- make sure you purified just your protein
Biochemical assay
Measure how much reactants and products you have
Give substrates and see if products are produced
Ways to detect proteins
- Optical density: Concentration of protein can be evaluated
- Gel electrophoresis
- Immunological methods (antibodies)
- Biochemical Assays
What various properties of a protein can be examined with gel electrophoresis?
Monomeric proteins: single subunit
Multimeric proteins: more than one subunit
Homodimers: dimeric proteins where both subunits are identical.
Heterodimers: dimeric proteins where both subunits aredifferent.
SDS Page
SDS polyacrylamide gel electrophoresis
Proteins are treated first with SDS and β-mercaptaethanol
SDS maintains the denatured conformation of proteins and coats the polypeptides with a negative charge, have an equivalent charge/mass ratio
β -mercaptoethanol reduces disulfide bonds
Process of SDS Page
Denature and coat with SDS
Place mixture of proteins on gel, apply electric field
Stain to visualize separated bands
How do you use protein specific stains?
You can detect the polypeptides in the gel
First you run the gel just to see if the protein is present, then you purify more and run the gel again. (Can’t use affinity column right away because if there is a lot of stuff it doesn’t work as well)
2-D Gel Electrophoresis
Protein samples containing a large number of proteins can be separated using electrophoresis in two dimensions
The first dimension is through a gel containing a pH gradient. Proteins migrate until they reach the pH at which they have no charge.
The second dimension is generally an SDS gel, which separates according to size
Separation in the first degree of 2D electrophoresis
Separation in the first dimension is based on isoelectric point (net 0 charge)of a protein. When it reaches this point, the protein stops migrating
- Charge on a protein is sum of all charges on amino acid side chains
- As the pH goes from low to high (4-10), amino acids change their ionization state
Where are the positive and negative electrodes in 2D gel electrophoresis
Electrophoresis in the first gel has the positive electrode on top and the negative electrode on the bottom
The reverse is true for electrophoresis through the SDS gel.
What makes proteins move according to pH?
high hydrogen content at low pH (acidic), + charges move to the basic side (high pH)
What is surprising about 2D
Can resolve more around 2000 proteins at once
2D gels can resolve proteins thatdiffer by a single unit of charge
Western blot
Can help you find one specific protein
Uses antibodies
Process of western blotting
Run an electric current through an SDS gel onto a nitrocellulose filter that binds to the proteins
Incubate with an antibody for that protein, then wash
Incubate with secondary chromagenic antibody that binds to the first antibodies (alkaline phosphatase)
Substrate is added that the secondary antibody cleaves and color shows where the protein is on the gel
ELISA
Cell culture plate
Well is coated with antigen
Add plasma from test individual. If antibody is present it binds to antigen
Secondary antibody binds to antibody in plasma (Alkaline phosphatase)
Test enzyme catalyzes a reaction that changes the color of the solution indicating presence of antibody
Home pregnancy tests
Antibody to Chorionic Gonadotropin on strip
Does urine contain CG?
Enzyme linked secondary antibody to CG that gives color reaction
immunofluorescence
Can detect proteins in cell
Kinesin (yellow)
Tubulin (green)
Overlap (orange)
Useful in cell biology to see where things actually occur in a cell and their arrangement
Edman degradation
- This technique allows sequential analysis of amino acids starting at the N-terminus
- Approximately 30 or so amino acids can be clearly determined.
- To sequence an entire protein, small sections have to be determined and then pieced together (constant washing with acid not good)
Process of Edman degradation
Phenylisothiocyante (PITC)- Binds to amino end of protein, label that first amino acid. Specific acid that recognizes pitc and it cleaves bind between first and second amino acid
Now you have a lot of the first amino acid in the solution, repeat.
Proteins can be broken into smaller peptides for sequence determination
- Start with a purified protein
- You can use proteases that cleave bonds between specific amino acid pairs
- These can then be subjected to Edman degradation.
- The entire protein sequence can be pieced together only after sequencing of two sets of protease fragments.
What order were they in? Cut different parts with second protease, line up AA by looking at overlapping fragments
Proteomics
- Study of all proteins produced in cell or species under a givenset of conditions.
- 2D gel electrophoresis coupled with mass spectrometry to identify MW of protein
