BICH 605. BICH 605; Fall 2009. October 6, 8, 20 & 22 Larry Dangott Department of Biochemistry and Biophysics Room 440 BioBio 845-2965 [email protected] BICH 605. OUTLINE. Planning : Method Development; Strategies Activity Tracking; Fraction ‘pooling’ Techniques :
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To present an OVERVIEW of techniques used in Protein Purification and Analysis.
It helps to know something about your protein
Source (organism; tissue; organelle; amount)
Multimer vs. Monomer
Affects buffer choices (assembly vs. disassembly)
Affects choice of separation media (size)
Cytosol vs. Membrane
Affects pre-fractionation choices (extraction)
Separation methods (centrifuge, columns)
Affects buffer choices (detergent)
Size (sort of related to Multimer vs. Monomer)
Affects choice of separation media (GFC)
Affects solubility (larger proteins like to precipitate)
Isoelectric point (pI)
Affects choice of separation media (charge)
Affects solubility (precipitate at pI)
Affects buffer choices (precipitation point; charge)
Affects choice of separation media (affinity)
Important Steps You May Use:
A SIMPLE STRATEGY
A COMPLEX STRATEGY FOR PROTEIN PURIFICATION
Systematic method development requires.....
Defining a way of quantifying, or at least identifying, the presence of your target molecule, and of assessing its purity.
Don’t rely solely on literature (or coworker) statements. Verify yourself. 50% success rate.
Keep a record of your purification process.
Notebook, notebook, notebook………. . . .
Our Example: Enzyme Purification
There are two major objectives in enzyme purification:
To obtain the highest SPECIFIC ACTIVITY possible, measured as activity per unit protein
To obtain the MAXIMUM YIELD of enzymatic activity. (Theoretically, this is 100%. Practically, one is usually happy to settle for something like 30%.)
When purifying a protein, one wants to keep track of how one is doing relative to the two major objectives.
Therefore, at each step, one must measure:
These measurements are combined in the calculation of:
Total activity = Enzyme activity/aliquot volume X Total volume
Total protein = Protein/aliquot volume X Total volume
Specific activity = Enzyme activity in an aliquot/Amt ofProtein in the aliquot (THIS IS THE BIG ONE)
(In measurements of total activity and protein, remember to adjust for volumes set aside for various reasons. If this is not done, the yield will be artificially low).
Use to calculate Specific Activity
Vol X Activity Units/vol = Total Activity Units
Vol X mg/ml = Total Protein (mg)
Divide Total Activity Units by Total Protein (mg) = Specific Activity in Units/protein (mg)
Fold purification goes UP
Yield goes DOWN
Divide current Specific Activity by Initial Specific Activity = Fold Purified
Divide current Total Activity Units by Original Activity Units = % Yield
Mutant Tyrosine Hydroxylase; Ion Exchange; NaCl Gradient
Pure Mutant TyrOH has a Vmax of ~12
Data courtesy of Colette Daubner; Fitzpatrick Lab
The less prevalent the protein is in the cytosol, the higher the degree of purification that will be required for its purification to homogeneity.
A protein that is 50% of the cellular protein needs to be purified only 2-fold.
A protein that is only 0.1% of the cellular protein needs to be purified 1000-fold.
A TYPICAL STRATEGY FOR PROTEIN PURIFICATION
Mode of monitoring the purification………………
Tracking your protein is critically important. How do you know where it is?
Biological Assay (usually specific; extremely sensitive; slower)
Binding Assay (usually specific; sensitive, semi-automate)
Chemical Assay (colorimetric assays, enzyme assays)
Physical Assay (mass spec, UV spectrometry)
Separation Assay (electrophoresis)
Electrophoresis is a electrically driven sieving process used to separate complex mixtures of proteins. Can be ANALYTICAL or PREPARATIVE.
SDS PAGE is used to investigate subunit composition and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further micro-analytical applications
Principle of SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
Most proteins bind the ionic detergent, SDS (sodium dodecyl sulfate), in a constant weight-to-detergent ratio, leading to identical negative charge density per mass for the denatured proteins and a uniform shape.
Thus, theoretically, SDS-protein complexes migrate through a solid matrix (polyacrylamide) and are separated according to size, not charge.
Polypeptide composition and fraction profiling:
Purified protein complexes or multimeric proteins consisting of subunits of different molecular size will be resolved into constituent polypeptides. Screen fractions during protein purification.
Quaternary structure profile:
Comparison of the protein bands obtained under non-reducing and reducing conditions provides information about the molecular size of subunits and protein complexes.
The relationship between the relative mobility and log molecular weight is linear over some range. With the use of plots like those shown here, the molecular weight of an unknown protein (or its' subunits) may be determined by comparison with known protein standards.
In SDS gel electrophoresis, negatively charged, SDS-coated proteins migrate in response to an electrical field through pores in a crosslinked polyacrylamide gel matrix
Pore size decreases with higher acrylamide concentrations
Smaller pores are used for smaller proteins/peptides; larger pore sizes are used for larger proteins.
Proteins to be analyzed are solubilized and denatured by boiling (or heating) in the presence of SDS and reducing reagent, an aliquot of the protein solution is applied to a gel lane, and the individual proteins are separated electrophoretically.
The reducing reagent β-Mercaptoethanol (-ME) or (dithiothreitol (DTT)) is added during solubilization to reduce disulfide bonds.
The polyacrylamide gel is cast as a separating gel (sometimes called the resolving or running gel) topped by a stacking gel and secured in an electrophoresis apparatus (see figure).
The stacking gel is run at slightly acid pH (6.8). The separating gel is run at pH 8.8. The stacking gel is ~4% acrylamide and the separating gel is a higher concentration.
The stacker brings the proteins to a common ‘starting line’ and the separator sieves them apart. The concentration of acrylamide in the separating gel is determined by the range of molecular weights of interest.
Tris-Glycine in Upper Buffer
Tris-HCl pH 6.8 in Stacking Gel
Tris-HCl pH 8.8 in Separating Gel
Formation of an ion front
It is the voltage gradient that sharpens the ion boundary
What happens to proteins?
In separating gel
Glycine mobility increases, becomes greater than protein mobility, but still slower than Cl-
Protein sample, now in a narrow band, encounters both the increase in pH and decrease in pore size.
Increase in pH would tend to increase electrophoretic mobility, but smaller pores decrease mobility.
Relative rate of movement of ions in separating gel is chloride > glycinate > protein.
Proteins separate based on charge/mass ratio and on size and shape parameters.
Coomassie Blue G-250 or R-250 staining 50 ng fixing
Silver 1 ng fixing
Fluorescent stains (Sypro) 10 ng non-fixing
Negative stains (zinc, copper) 1- 10 ng non-fixing
Relative Mobility (Rf)SDS PAGE
MW ESTIMATION BY SDS-PAGE IS ONLY APPROXIMATE AND IS REFERRED TO AS APPARENT MOLECULAR WEIGHT. Unusual protein compositions or physical properties can cause anomalous mobilities during SDS-PAGE.
SDS gels can be used as a micro-purification step and the individual polypeptides can be isolated from the gel by electroelution or electroblotting and the amino acid sequences can be determined or peptide maps obtained.
Isoelectric Point (pI) is specific pH at which net charge equals zero
IEF is a technique to separate proteins
based on Isoelectric Point (native or denatured)
At pI, protein has no net charge and will not migrate in an electric field
IEF CAN BE PERFORMED WITH MOBILE pH GRADIENTS OR IMMOBILIZED pH GRADIENTS
Mobile gradients are prepared with Carrier Ampholytes (CAs) (mixed polymers (300-1000 Da in size) mixed with solid support (mobile).
Immobile gradients are prepared by covalently coupling Ampholytes to solid support and blending.
Solid support is usually polyacrylamide but can be agarose for preparative purposes
MOBILE pH GRADIENT IEF
IMMOBILIZED pH GRADIENT IEF
Combine IEF & SDS PAGE
Zoom gels (pH range)