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GMS BI 555/755 Lecture 3: Techniques for Protein Characterization. Reading: Berg/Stryer, 6 th Ed. Chapter 3 Protein extraction, sub-cellular fractionation Size-based separations Dialysis Gel filtration Ultracentrifugation Charged-based separations Ion Exchange chromatography

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Gms bi 555 755 lecture 3 techniques for protein characterization
GMS BI 555/755 Lecture 3: Techniques for Protein Characterization

  • Reading: Berg/Stryer, 6th Ed. Chapter 3

  • Protein extraction, sub-cellular fractionation

  • Size-based separations

    • Dialysis

    • Gel filtration

    • Ultracentrifugation

  • Charged-based separations

    • Ion Exchange chromatography

  • Affinity chromatography

  • Reversed phase chromatography

  • Electrophoresis

    • SDS-PAGE

    • Isoelectric focusing

    • 2D Gel electrophoresis

  • Circular dichroism

  • Amino acid analysis

  • Edman degradation

  • Peptide synthesis

  • Proteomics

Protein: from Greek πρώτα ("prota"), meaning "of primary importance"

Protean: readily assuming different forms or characters; extremely variable

GMS BI 555/755 Lecture 3.


Proteins must be extracted from their cell or tissue context for purification
Proteins must be extracted from their cell or tissue context for purification

  • Classical and contemporary reasons for sub-cellular fractionation

  • Yield, activity, specific activity

  • Differential Centrifugation. Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will form a pellet at lower centrifugal force than will the less-dense material. The isolated fractions can be used for further purification.

  • Alternatives: cells may be disrupted by treatment with low or high salt (osmotic lysis) and the protein in question purified from the cytosol or membrane fraction.

  • Selective precipitation according to protein class

    • Ammonium sulfate ppt

    • NaCl ppt

    • Ethanol precipitation of ECM proteins

  • Enzyme activity

  • Patterns of protein expression

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GMS BI 555/755 Lecture 3.


Size based separations dialysis
Size based separations: dialysis for purification

  • Centrifugal concentrators:

  • Centrifugal force pushes solution through a membrane with size selective pores

  • Useful for concentrating, desalting proteins

  • Also dialysis concentrator cells

  • Dialysis. Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.

  • Separation of large from small molecules

  • Solubility, recovery serious issues

GMS BI 555/755 Lecture 3.


Size based separations gel filtration chromatography
Size based separations: Gel-filtration chromatography for purification

  • AKA size exclusion chromatography

  • Smaller proteins experience a higher mobile phase volume because they are able to enter pores of the stationary phase beads.

  • The elution volume is inversely related to the molecular weight

  • Chromatographic resolution depends on a number of factor including bead diameter, pore size, salt concentration, column volume, flow rate

  • Other factors constant, resolution increases with column volume

  • Protein must be freely soluble

  • Detergents necessary for membrane proteins

  • Typically 0.15 M salt necessary to prevent non-specific interactions between proteins and beads.

  • Volume of protein injected onto column must be <2% of column volume.

  • Eluant is diluted

GMS BI 555/755 Lecture 3.


Gel filtration size exclusion
Gel filtration/Size exclusion for purification

Gel filtration: principles and methods, GE Healthcare

GMS BI 555/755 Lecture 3.


Gel filtration size exclusion1
Gel filtration/Size exclusion for purification

Gel filtration: principles and methods, GE Healthcare

GMS BI 555/755 Lecture 3.


Size based separations gel filtration chromatography1
Size based separations: Gel-filtration chromatography for purification

  • Gel filtration chromatography of a protein mixture

  • thyroglobulin (669 kd),

  • catalase (232 kd),

  • bovine serum albumin (67 kd),

  • ovalbumin (43 kd), and (

  • ribonuclease (13.4 kd).

GMS BI 555/755 Lecture 3.


Gel filtration size exclusion sample volume
Gel filtration/Size exclusion: sample volume for purification

Gel filtration: principles and methods, GE Healthcare


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Log scale for purification

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SEC: optimization of flow rate for purification

Van Deemter equation

A = Eddy-diffusion

B = Longitudinal diffusion

C = mass transfer kinetics of the analyte between mobile and stationary phase

u = Linear Velocity.

48 Da

500 Da

1000 Da

Ziegler, A.; Zaia, J. J Chromatogr B Analyt Technol Biomed Life Sci 2006, 837, 76-86.

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Protein analysis by ultracentrifugation for purificationSub-cellular fractionation (preparative)Analysis of protein mass, density, shape, binding (analytical)

  • A particle will move through a liquid medium when subjected to a centrifugal force. A convenient means of quantifying the rate of movement is to calculate the sedimentation coefficient, s, of a particle by using the following equation:

  • where

  • m is the mass of the particle,

  • is the partial specific volume (the reciprocal of the particle density),

    ρ is the density of the medium and

    f is the frictional coefficient (a measure of the shape of the particle).

    The (1 - ρ) term is the buoyant force exerted by liquid medium.

  • Sedimentation coefficients are usually expressed in Svedberg units (S), equal to 10-13 s. The smaller the S value, the slower a molecule moves in a centrifugal field.

GMS BI 555/755 Lecture 3.


Protein analysis by ultracentrifugation
Protein analysis by ultracentrifugation for purification

1. The sedimentation velocity of a particle depends in part on its mass. A more massive particle sediments more rapidly than does a less massive particle of the same shape and density.2. Shape, too, influences the sedimentation velocity because it affects the viscous drag. The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass. Hence, elongated particles sediment more slowly than do spherical ones of the same mass.3. A dense particle moves more rapidly than does a less dense one because the opposing buoyant force (1 - ρ) is smaller for the denser particle.4. The sedimentation velocity also depends on the density of the solution. (ρ). Particles sink when ρ < 1, float when ρ > 1, and do not move when ρ = 1.

Density and Sedimentation Coefficients of Cellular Components. [After L. J. Kleinsmith and V. M. Kish, Principles of Cell and Molecular Biology, 2d ed. (Harper Collins, 1995), p. 138.]

GMS BI 555/755 Lecture 3.


Protein analysis by ultracentrifugation1
Protein analysis by ultracentrifugation for purification

5% sucrose 25% sucrose

Zonal Centrifugation. The steps are as follows: (A) form a density gradient, (B) layer the sample on top of the gradient, (C) place the tube in a swinging-bucket rotor and centrifuge it, and (D) collect the samples. [After D. Freifelder, Physical Biochemistry, 2d ed. (W. H. Freeman and Company, 1982), p. 397.]

  • Protein mass can be measured accurately by sedimentation equilibrium

  • Mass measurement under native conditions, applicable to multiprotein complexes

GMS BI 555/755 Lecture 3.


Charge based separations ion exchange chromatography
Charge-based separations: ion exchange chromatography for purification

Weak Anion exchange (WAX)

Weak Cation exchange (WCX)

Strong cation exchange (SCX)

Sulfonic acid functional groups

Strong anion exchange (SAX)

Quaternary amino ethyl (QAE)

  • A protein in a buffer at pH > pKa will be negatively charged and able to bind an anion exchange resin.

  • A protein in a buffer at pH < pKa will be positively charged and able to bind a cation exchange resin

  • Proteins may be eluted with a gradient of increasing salt (NaCl, or other)

  • The elution order will depend on how tightly (charged) is a given protein

  • Injection volume not limited

GMS BI 555/755 Lecture 3.


Charge based separations ion exchange chromatography1
Charge-based separations: ion exchange chromatography for purification

  • Ion exchange entails loading a protein mixture at a given pH in a low salt concentration buffer and washing the unbound material through the column

  • For cation exchange, positively charged proteins bind to the column, negatively charged proteins pass through

  • For anion exchange, negatively charged proteins bind the column, positively charged ones pass through

  • Bound proteins are eluted by a gradient of increasing salt concentration

  • Weakly bound proteins elute at a relatively low salt concentration, tightly bound ones at higher salt concentration

  • Proteins are focused by binding to the column. Thus, large volumes of solution may be applied to the column.

GMS BI 555/755 Lecture 3.


Affinity chromatography
Affinity Chromatography for purification

  • A protein may be purified based on its binding to a chemical group or another protein

  • Immobilize the ligand on a chromatographic bead (many standard chemistries available)

  • Pass the protein mixture through the column

  • Wash non-bound material through the column

  • Elute the bound proteins either using a competing ligand, an increasing salt concentration, or other denaturatn

  • Examples

    • Avidin-biotin

    • Poly-His tags binding to immobilized metal columns

    • Protein A/G binding to antibodies

    • Growth factor binding to heparin columns

    • Transcription factor binding to immobilized DNA seq

    • Purification of recombinant fusion proteins (GST, GFP, FLAG)

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High performance liquid chromatography
High performance liquid chromatography for purification

  • Chromatographic resolution increases as the size of the chromatographic beads decreases

  • The pressure required to push solvent (mobile phase) through the packed beads increases as their size decreases

  • High performance liquid chromatography (HPLC): a pumping system that pushes solvent through a column packed with small beads (approximately 5 microns).

  • All tubing, columns, made of stainless steel to withstand pressures up to 2500 psi or so.

  • Higher performance and cost than conventional low pressure liquid chromatography.

  • Typically one begins with low pressure chromatography to concentrate the protein and then switches to HPLC for later steps

  • Available with any chromatography mode

  • Many models and manufacturers

GMS BI 555/755 Lecture 3.


Reversed phase high performance liquid chromatography
Reversed phase high performance liquid chromatography for purification

  • Reversed phase HPLC is a ubiquitously useful means of separating proteolytic peptides using a gradient from low to high percent organic

  • Peptides bind to C18 stationary phase in low organic solvent, elute with a gradient of increasing organic content

  • Trifluoroacetic acid (TFA) is an ion-pairing agent that prevents charge-based interactions between peptides and stationary phase

  • Proteins are denatured by TFA and organic

  • Useful as a means for separating intact proteins for structural studies.

Absorbance 214 nm

% acetonitrile

Time

Reversed phase separation of a tryptic digestion of apotransferrin. Gradient of 0-50% acetonitrile, 0.1% trifluoroacetic acid over 100 min,

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)protein purity, homogeneity, distribution

Thin gel, molecular sieve

  • Proteins prepared by boiling in an SDS-buffer in the presence of a reductant (mercaptoethanol, DTT)

  • Protein solution loaded into a gel lane

  • Applied electric field causes protein-SDS complexes to migrate to the anode (positively charged) electrode at the bottom

  • Proteins separated approximately based on size as they migrate through the porous polyacrylamide gel matrix

ν =Ez/f

ν = velocity

E = elec. field str.

z = net prot charge

f = frictional coeff.

Polyanionic

~1 SDS/2AA

GMS BI 555/755 Lecture 3.


Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Chromatographic fractions

Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDS-polyacrylamide gel can be visualized by staining with Coomassie blue.

  • Formation of cross-linked polyacrylamide gel.

  • Many recipes for pouring gradient gels for protein separations

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page1
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

  • Protein-SDS complexes migrate approximately according to mass

  • Mobility proportional to the logarithm of mass

  • This assumes that the ratio of bound SDS molecules per mass unit of protein is the same for all proteins

  • In reality, this ratio, and thus the relative mobility in the electric field, varies according to several factors

    • Hydrophobicity of polypeptide (membrane proteins migrate differently than soluble proteins)

    • Glycosylation: most proteins are glycosylated, and many glycans are acidic

    • Phosphorylation: ubiquitous signalling mechanism, lowers pI

    • Mature protein heterogeneity results in diffuse bands.

  • Migration proportional to log(mass)

  • Can resolve 2% difference in mass

  • Resolution = m/Δm = ~50

Log scale

GMS BI 555/755 Lecture 3.


Two dimensional gel electrophoresis

Cytochrome C pI 10.8 (SDS-PAGE)

Serum albumin pI 4.8

Two-dimensional gel electrophoresis

  • IEF

  • Isoelectric point = pI = pH where z = 0

  • Loading an issue

The Principle of Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation.

Two-Dimensional Gel Electrophoresis. (A) A protein sample is initially fractionated in one dimension by isoelectric focusing. The isoelectric focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is performed in the second dimension, perpendicular to the original separation. Proteins with the same pI are now separated on the basis of mass. (B) Proteins from E. coli were separated by two-dimensional gel electrophoresis, resolving more than a thousand different proteins. The proteins were first separated according to their isoelectric pH in the horizontal direction and then by their apparent mass in the vertical direction.

>1000 spots stained

i.d. a problem

GMS BI 555/755 Lecture 3.


2d electrophoresis

Advantages (SDS-PAGE)

Separation and visualization of 1000’s of proteins in two dimensions based on pI and molecular weight

Able to visualize protein pattern easily

Comparison of protein expression patterns as a function of biological change

Many commercial 2DE systems available, chemistries well worked out

Disadvantages

Dynamic range (limited protein loading)

Visualizes the most abundant proteins; low abundance proteins not detected

Proteins must be excised from gels for identification using Edman degradation or mass spectrometry

Difficult to get reproducible separation; alignment of different gels requires sophisticated software

Most animal proteins are post-translationally modified and are observed as more than one spot.

2D electrophoresis

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Circular dichroism and protein structrure (SDS-PAGE)

Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.

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Circular dichroism for protein structure analysis: characteristic profiles according to secondary structure.

Lesk: Introduction to Protein Science, chap 3, Fig. 11

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CD: fixed wavelength measurement for determination of protein melting point (protein stability)

Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.

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  • Use of CD to assess the folded state of a protein: protein melting point (protein stability)

  • A properly folded protein has CD signal in both near and far UV regions

  • A molten globule protein has secondary, not tertiary structure, and lacks near-UV signal

Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.

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Amino acid analysis

  • Is the protein pure? protein melting point (protein stability)

  • Is it the right protein?

  • Does it have the expected AA composition?

Amino acid analysis

nm

  • Amino acid analysis involves four basic steps:

  • Hydrolyze a protein to individual constituent amino acids (6N HCl)

  • Label amino acids with a detectable UV-absorbing or fluorescent marker

  • Separate different types of amino acids by chromatography

  • Measure relative amounts of each amino acid type based on intensity of the detectable marker associated with the emergence of each type of amino acid from the chromatographic system

  • Requires a few hundred picomoles of purified protein

GMS BI 555/755 Lecture 3.


Amino acid analysis1
Amino acid analysis protein melting point (protein stability)

Fluorescamine reacts with the α-amino group of an amino acid to form a fluorescent derivative.

Ala-GlyAsp-Phe-Arg-Gly

Determination of Amino Acid Composition. Different amino acids in a peptide hydrolysate can be separated by ion-exchange chromatography on a sulfonated polystyrene resin (such as Dowex-50). Buffers (in this case, sodium citrate) of increasing pH are used to elute the amino acids from the column. The amount of each amino acid present is determined from the absorbance. Aspartate, which has an acidic side chain, is first to emerge, whereas arginine, which has a basic side chain, is the last. The original peptide is revealed to be composed of one aspartate, one alanine, one phenylalanine, one arginine, and two glycine residues.

GMS BI 555/755 Lecture 3.


Amino acid analysis2
Amino acid analysis protein melting point (protein stability)

  • Useful method for establishing that a given protein preparation has an amino acid composition that matches its theoretical sequence

  • Used in industry for recombinant protein-based products

    • Recombinant drugs

    • Antibodies

    • Animal feeds and additives

  • Requires a relatively large quantity of pure protein, primarily due to limitations of the hydrolysis step (high temperature, 6N HCl)

    ~300 pmol or 30 μg of a 100 kDa protein

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Edman Degradation: N-terminal protein sequence analysis protein melting point (protein stability)Pehr Edman, 1950, automated protein sequencer, 1967

  • Phenylisothiocyanate (PITC) reacts with protein N-terminus. Under mild acid conditions to form a phenylthiohydantoin (PTH) amino acid derivative

  • The reaction may be repeated, one cycle per AA residue

  • PTH-AAs detected using chromatography

  • Much engineering and optimization of reaction and detection system

GMS BI 555/755 Lecture 3.


Edman degradation
Edman Degradation protein melting point (protein stability)

The labeled amino-terminal residue (PTH-alanine in the first round) can be released without hydrolyzing the rest of the peptide. Hence, the amino-terminal residue of the shortened peptide (Gly-Asp-Phe-Arg-Gly) can be determined in the second round. Three more rounds of the Edman degradation reveal the complete sequence of the original peptide.

GMS BI 555/755 Lecture 3.


Edman degradation1
Edman Degradation protein melting point (protein stability)

Separation of PTH-Amino Acids. PTH-amino acids can be rapidly separated by high-pressure liquid chromatography (HPLC). In this HPLC profile, a mixture of PTH-amino acids is clearly resolved into its components. An unknown amino acid can be identified by its elution position relative to the known ones.

GMS BI 555/755 Lecture 3.


Edman degradation2

Advantages: protein melting point (protein stability)

Relatively straight-forward interpretation based on chromatographic retention time

Automated gas phase Edman sequencers available (?)

Able to handle 2 or 3 component mixtures in favorable cases

Absolute quantification of released amino acids

Can analyze PTM-modified AAs

Disadvantages

Slow, ~1 hr per cycle

Peptides must be pure, not able to handle complex mixtures

Blocked N-terminus prevents Edman degradation (Acetylation, pyroglutamic acid, others)

Used primarily for analysis of protein-based pharmaceutical products

Heroic effort necessary to completely sequence a protein

Lack of market for commercial instruments

Edman Degradation

GMS BI 555/755 Lecture 3.


Cleavage of proteins at specific amino acid residues peptide mapping
Cleavage of proteins at specific amino acid residues: protein melting point (protein stability)Peptide mapping

  • Met is a rare AA residue

  • Cyanogen bromide cleavage generates large peptides (membrane proteins)

Cleavage by Cyanogen Bromide. Cyanogen bromide cleaves polypeptides on the carboxyl side of methionine residues

  • Lys and Arg are relatively common AA residues

  • Trypsin digestion reliably cleaves to the C-terminal side for denatured proteins

  • Produces peptides ranging approximately from 500-2500 Da (many exceptions)

Cleavage by Trypsin. Trypsin hydrolyzes polypeptides on the carboxyl side of arginine and lysine residues

GMS BI 555/755 Lecture 3.


Reliable methods for specific protein cleavage
Reliable methods for specific protein cleavage protein melting point (protein stability)

  • Enzymatic

    • Trypsin (C-term of Lys, Arg)

    • Lys-C (C-term of Lys)

    • Asp-N (N-term of Asp)

    • Chymotrypsin (Tyr, Trp, Phe, Leu, Met, others)

    • (Other enzymes less reliable)

  • Chemical

    • Cyanogen bromide

    • (Other chemical methods less reliable)

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Informational value of amino acid sequences
Informational value of amino acid sequences protein melting point (protein stability)

  • BLAST (Basic Local Alignment Search Tool) www.ncbi.nih.gov computes alignments for a given sequence to known sequences

  • Does a protein belong to a given family?

  • Evolutionary trees based on protein sequence similarity

  • Are there internal repeats in a protein sequence? Evidence for duplication of primordial genes

  • Sequence data may be used to generate antibodies against a protein of interest (Western blotting, immunohistochemistry, immunoprecipitation, ELISA)

  • AA sequences useful for design of DNA probes specific to the corresponding gene.

GMS BI 555/755 Lecture 3.


Solid phase peptide synthesis
Solid phase peptide synthesis protein melting point (protein stability)

  • Amino Acid Activation. Dicyclohexylcarbodiimide is used to activate carboxyl groups for the formation of peptide bonds.

  • Solid phase peptide synthesis starts by immobilizing the C-terminal amino acid of the target peptide sequence on a resin

  • The t-Boc protecting group is removed

  • The second t-Boc amino acid is then added and coupled using carbodiimide chemistry

  • Carbodiimide reacts with and activates the carboxyl group of the t-Boc-AA to facilitate peptide bond formation

Berg

GMS BI 555/755 Lecture 3.


Solid phase peptide synthesis1
Solid phase peptide synthesis protein melting point (protein stability)

The first amino acid (blue) of the desired peptide is attached at its carboxyl end by esterification to a polystyrene bead. The amino group of this amino acid is blocked by the attachment of a tertbutyloxycarbonyl (tBOC) group (red), which is removed by treatment with trifluoroacetic acid (CF3COOH). The resulting free amino group forms a peptide bond with a second amino acid, which is presented with a reactive carboxyl group and a blocked amino group, together with the coupling agent dicyclohexylcarbodiimide (DCC). The process is repeated until the desired product is obtained; the peptide is then chemically cleaved from the bead with hydrofluoric acid (HF). [See R. B. Merrifield, L. D. Vizioli, and H. G. Boman, 1982, Biochemistry21:5020.

Lodish

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Elisa enzyme linked immunosorbent assay
ELISA: Enzyme-linked Immunosorbent Assay protein melting point (protein stability)

Immobilization: physical or chemical?

Indirect ELISA and Sandwich ELISA (A) In indirect ELISA, the production of color indicates the amount of an antibody to a specific antigen. (B) In sandwich ELISA, the production of color indicates the quantity of antigen. [After R. A. Goldsby, T. J. Kindt, B. A. Osborne, Kuby Immunology, 4th ed. (W. H. Freeman and Company, 2000), p. 162.]

GMS BI 555/755 Lecture 3.


Elisa

Advantages protein melting point (protein stability)

Extremely sensitive detection of Ab or Ag in question

Amenable to high-throughput multiwell format

Quantitative readout of Ag or Ab level in a fluid

Many fluorescent tags available for ELISA detectionn (radiolabeling)

Many commercial kits for ELISA development, detection, etc.

Disadvantages

Requires high quality Abs to each Ag to be tested; time consuming and expensive to generate Abs (requires high affinity, specific Abs)

Generation of high quality Abs is an inexact science

ELISA conditions (buffer salt concentrations, additives, detergents, washing protocolls, etc) are specific to each Ag-Ab interaction; it is difficult to make a single assay for more than one Ag-Ab pair.

ELISA

GMS BI 555/755 Lecture 3.


Western blotting
Western Blotting protein melting point (protein stability)

  • Western Blotting. Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained with radioactive (or fluorescently labeled) antibody. A band corresponding to the protein to which the antibody binds appears in the autoradiogram.

  • Able to detect presence of a given protein in a complex cell lysate

  • Semi-quantitative measurement of protein level

  • Depends on transfer of proteins from gel to membrane

  • Not all abs have high enough affinity to be useful for Western blotting

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Genomics and proteomics
Genomics and Proteomics protein melting point (protein stability)

  • Genomics: organismal study of patterns of gene expression related to disease and developmental processes

    • Human genome project: all human genes sequenced

    • Approximately 30k human genes

  • Functional genomics: effort to make use of the vast wealth of data from the various genomics projects to understand gene and protein functions and interactions

    • Focusses on dynamic aspects of gene transcription, translation, and protein-protein interaction

  • Proteomics: large scale study of protein expression: functions and structures

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Why study protein expression? protein melting point (protein stability)

(the steps of gene expression control)

Nucleus

Cytosol

RNA

degradation

control

inactive

mRNA

primary

RNA

transcript

DNA

mRNA

mRNA

translation

control

transcriptional

control

RNA

processing

control

RNA

transport

control

modified

protein

protein

  • Consequence: protein expression is usually poorly correlated to mRNA expression level

    • Gygi, et al., Mol. Cell. Biol., 1999, 19, p. 1720-1730)

post-

translational

control

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Some common post translational modifications of proteins
Some common post-translational modifications of proteins protein melting point (protein stability)

Mann, M., and Jensen, O. N. (2003). Nat Biotechnol21, 255-61.

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