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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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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]


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BICH 605

OUTLINE

Planning:

  • Method Development; Strategies

  • Activity Tracking; Fraction ‘pooling’

    Techniques:

  • Electrophoresis (SDS, Isoelectric Focusing)

  • Chromatography (GFC, IEX, Affinity, rpHPLC)

  • Structural Characterization (Amino Acid Analysis; Protein Sequencing)

  • Proteomics (Protein ID and characterization using mass spectrometry)

    To present an OVERVIEW of techniques used in Protein Purification and Analysis.


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Protein Purification

It helps to know something about your protein

  • Source (organism; tissue; organelle; amount)

  • Assemblage vs. monomer

  • Cytosolic vs. membrane-bound

  • Size

  • Isoelectric point (pI)

  • Post-translational modification

  • Relative abundance


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Protein Purification

Source (organism; tissue; organelle; amount)

  • Natural vs. Recombinant (tagged?)

  • Tissue (bone (hard), blood (liquid), heart (soft), brain (fatty)); extraction

  • Organelle (nucleus, mitochondria, ER, plasma membrane); pre-fractionation

  • Amount (a LOT or a little; scale); cost and practicality (myoglobin = easy; EGFR = hard)


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Protein Purification

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)

Post-Translational Modification

Affects choice of separation media (affinity)


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Protein Purification

Important Steps You May Use:

  • Extraction (French press, sonication, detergent, homogenization)

  • Centrifugation (low speed, ultra-speeds, differential gradient) Protein estimation method (colorimetric, spectroscopy)

  • Protein concentrating method (salt or organic precipitation, lyophilization, membrane filtration)

  • Chromatography (IEX, gel filtration, chromatofocusing)

  • Electrophoresis (IEF, preparative native or SDS)


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A SIMPLE STRATEGY

His-tag: affinity

Protein Purification

A COMPLEX STRATEGY FOR PROTEIN PURIFICATION

Sample Preparation

  • Extraction (grinding, detergent lysis, sonication)

  • Salt exchange (gel filtration, filters, dialysis)

    Capture

  • Ion Exchange

  • Affinity

  • Hydrophobic Interaction

    Intermediate Purification

  • Ion exchange

  • Hydrophobic Interaction

    Polishing

  • Gel Filtration

  • Reversed phase


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Happy Boss

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………. . . .


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Protein Purification

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%.)


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Protein Purification

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:

  • Volume

  • Protein concentration (colorimetric assay, UV)

  • Enzyme activity (units/ml; specific to ‘your’ protein)


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Protein Purification

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).


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Calculate Activity Units and Total Protein

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)


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KNOWING WHICH FRACTIONS TO POOL IS IMPORTANT

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


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Selecting Fractions based on Specific Activity and SDS PAGE

Mutant Tyrosine Hydroxylase; Ion Exchange; NaCl Gradient

RNA?

Pool

Stable?

Pure Mutant TyrOH has a Vmax of ~12

Data courtesy of Colette Daubner; Fitzpatrick Lab


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Protein Purification

The less prevalent the protein is in the cytosol, the higher the degree of purification that will be required for its purification to homogeneity.

For example:

A protein that is 50% of the cellular protein needs to be purified only 2-fold.

In contrast:

A protein that is only 0.1% of the cellular protein needs to be purified 1000-fold.


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Protein Purification

A TYPICAL STRATEGY FOR PROTEIN PURIFICATION

  • Sample Preparation

    • Extraction (grinding, detergent lysis, sonication)

    • Salt exchange (gel filtration, filters, dialysis)

  • Capture

    • Ion Exchange

    • Affinity

    • Hydrophobic Interaction

  • Intermediate Purification

    • Ion exchange

    • Hydrophobic Interaction

  • Polishing

    • Gel Filtration

    • Reversed phase

      Mode of monitoring the purification………………


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Protein 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)


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SDS PAGE ELECTROPHORETIC ANALYSIS OF PROTEINS

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.


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SDS PAGE

APPLICATIONS

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.

Size estimation:

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.  


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SDS PAGE

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.


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SDS PAGE

PROCEDURE

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.


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SDS PAGE

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.


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SDS PAGE

Tris-Glycine in Upper Buffer

Tris-HCl pH 6.8 in Stacking Gel

Tris-HCl pH 8.8 in Separating Gel


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SDS PAGE

Glycine equilibria


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SDS PAGE

Formation of an ion front


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SDS PAGE

It is the voltage gradient that sharpens the ion boundary


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SDS PAGE

What happens to proteins?


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SDS PAGE

In separating gel

Glycine mobility increases, becomes greater than protein mobility, but still slower than Cl-


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SDS PAGE

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.


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SDS PAGE

PROTEIN DETECTION

Detection limitFixing?

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

Sypro Ruby

Coomassie Blue

Silver


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Molecular Weight (Log Scale)

Relative Mobility (Rf)

SDS PAGE

SIZE ESTIMATION

IMPORTANT

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.


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ISOELECTRIC FOCUSING

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


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Isoelectric Focusing

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


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Isoelectric Focusing

MOBILE pH GRADIENT IEF

IMMOBILIZED pH GRADIENT IEF

  • ADVANTAGES of IMMOBILIZED GRADIENTS

  • Stable pH gradients

  • Ease of handling

  • Reproducibility

  • Extreme pH resolution


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2 Dimensional Gel Electrophoresis

Combine IEF & SDS PAGE

High Resolution

Zoom gels (pH range)

Detect isoforms

Post-translational modifications

Expression Proteomics


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2 Dimensional Gel Electrophoresis

  • Combine IEF & SDS PAGE

  • High Resolution

  • Zoom gels (pH range)

  • ANALYTICAL

  • Detect isoforms

  • Post-translational modifications

  • PREPARATIVE

  • Mass spectrometry



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