Isolation and quantification of Isolation and quantification of plant total proteinplant total protein

Dongping LuDongping Lu

ABE Workshop 2006ABE Workshop 2006

ABE Workshop ABE Workshop June 20 2006June 20 2006 Dongping LuDongping Lu

The detection of GFP and PDI2 at The detection of GFP and PDI2 at different levelsdifferent levels

DNA

RNA

Protein

In vivo and In situ

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Western blottingWestern blotting

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Protein isolationProtein isolation

Total proteinTotal protein Protein in different tissuesProtein in different tissues Organelle proteinOrganelle protein Membrane proteinMembrane protein Protein with different solubilityProtein with different solubility

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How to isolate total protein How to isolate total protein from plantfrom plant

Lyse the plant cell, Lyse the plant cell, Solubilze the proteins:Solubilze the proteins: To Solubilze To Solubilze membrane membrane

proteinprotein, we have to use , we have to use detergentsdetergents in the protein in the protein extraction bufferextraction buffer

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The often used detergents in the protein The often used detergents in the protein extraction bufferextraction buffer

Nonionic detergentsNonionic detergents (milder)(milder) Triton X-100Triton X-100: break lipid-lipid interaction and : break lipid-lipid interaction and lipid-protein interactionlipid-protein interaction

Anionic detergents Anionic detergents (more denaturing)(more denaturing) SDSSDS: protein-protein interaction: protein-protein interaction Sodium DeoxycholateSodium Deoxycholate: protein-protein interaction: protein-protein interaction

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Proteases inhibitorsProteases inhibitors Upon lysis of the cell, Upon lysis of the cell, proteasesproteases are released are released

into the lysateinto the lysate What are proteases?What are proteases? Where are the proteases from when isolating Where are the proteases from when isolating

the protein?the protein?

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What are proteases?What are proteases?

Protease: Protease: (proteinases(proteinases, , peptidasespeptidases or or proteolytic enzymesproteolytic enzymes) are enzymes that break ) are enzymes that break peptide bonds between amino acids of proteins peptide bonds between amino acids of proteins

Classes of proteolytic enzymesClasses of proteolytic enzymes::

Serine proteasesSerine proteases

AspartateAspartate proteasesproteases

Cysteine proteasesCysteine proteases

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Where are the proteases from when Where are the proteases from when isolating the protein?isolating the protein?

Animal cells: Lysosomes, contain a large variety of hydrolytic enzymes that degrade proteins and other substances

Plant cells: Vacuole, many hydrolytic enzymes found in vacuole resemble those present in Lysosomes of animal cells

other organelles also have proteases

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How to prevent the proteins from How to prevent the proteins from degradation by protease?degradation by protease?

the protein isolation is carried out at the protein isolation is carried out at low low temperaturetemperature to minimize the activities of to minimize the activities of these proteasesthese proteases

To further optimize the results, we use the To further optimize the results, we use the proteases inhibitorsproteases inhibitors

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Protease inhibitorsProtease inhibitors

Proteins: with domains that enter or block a Proteins: with domains that enter or block a protease active site to prevent substrate access, protease active site to prevent substrate access,

e.g. e.g. Cystatins. Cystatins.     Chemicals: some are used in the protein Chemicals: some are used in the protein

extraction bufferextraction buffer

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Often used chemical protease inhibitors Often used chemical protease inhibitors in protein isolationin protein isolation

EDTA (or EGTA)EDTA (or EGTA): chelating the : chelating the Ca2+,Ca2+,

PMSFPMSF: a general serine protease : a general serine protease inhibitor. It is the most common inhibitor. It is the most common inhibitor used in protein inhibitor used in protein purification. Soluble in purification. Soluble in isopropanol. isopropanol.

The The protease inhibitorsprotease inhibitors cocktail cocktail: : a mixture of several protease a mixture of several protease inhibitors with broad specificity for inhibitors with broad specificity for the inhibition of serine, cysteine, the inhibition of serine, cysteine, aspartic and aminopeptidases aspartic and aminopeptidases

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The protein quantificationThe protein quantification

UV 280 absorption UV 280 absorption ::

Colorimetric methodsColorimetric methods::

BiuretBiuret

Lowry Lowry

BradfordBradford

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UV absorption methodUV absorption method

The amino acids The amino acids tryptophan, tyrosine and tryptophan, tyrosine and phenylalanine absorb light phenylalanine absorb light in the in the UV wavelengthUV wavelength

Since the absorption is Since the absorption is proportional to proportional to concentration, this is a concentration, this is a useful way to quantitates useful way to quantitates protein concentration (for protein concentration (for proteins containing Trp) proteins containing Trp)

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Disadvantages of UV absorption Disadvantages of UV absorption methodmethod

If some proteins do not contain these amino acids, If some proteins do not contain these amino acids, it will not absorb UV light, it will not absorb UV light,

Nucleic acids (DNA, RNA) contaminant will also Nucleic acids (DNA, RNA) contaminant will also absorb UV light, absorb UV light,

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Colorimetric methodsColorimetric methods we can modify the protein sample with appropriate we can modify the protein sample with appropriate

reagents so as to produce a reagents so as to produce a color reactioncolor reaction and and measure protein concentration using a measure protein concentration using a spectrophotometer. spectrophotometer.

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Advantages of Colorimetric methodsAdvantages of Colorimetric methods

1. Cheap lamp! (tungsten light bulb versus 1. Cheap lamp! (tungsten light bulb versus deuterium for UV) deuterium for UV)

2. Cheap cuvette! (cheap glass or plastic 2. Cheap cuvette! (cheap glass or plastic versus quartz) versus quartz)

3. Not contaminating absorbance from nucleic 3. Not contaminating absorbance from nucleic acids! acids!

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Colorimetric methods I: Bradford MethodColorimetric methods I: Bradford Method

A dye known as A dye known as Coomassie Brilliant BlueCoomassie Brilliant Blue was was developed by the textile industry. It was developed by the textile industry. It was noticed to stain skin as well as the textiles. noticed to stain skin as well as the textiles.

Thus, this dye (which normally absorbs at Thus, this dye (which normally absorbs at 465nm) was known to bind to proteins and to 465nm) was known to bind to proteins and to absorb strongly at 595nm. absorb strongly at 595nm.

The assay is sensitive, but somewhat non-The assay is sensitive, but somewhat non-linear linear

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Colorimetric methods II:Colorimetric methods II: Biuret Biuret Under high pH (alkaline) Under high pH (alkaline)

conditions the conditions the copper II ioncopper II ion (Cu2+) is believed to form a (Cu2+) is believed to form a complex with complex with peptide nitrogens of peptide nitrogens of proteinsproteins::

This complex absorbs light at This complex absorbs light at 550nm550nm

the absorption is relatively weak, the absorption is relatively weak, thus, the method is somewhat thus, the method is somewhat insensitive and requires a insensitive and requires a relatively high concentration of relatively high concentration of protein protein

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Lowry MethodLowry Method reactivity of the peptide reactivity of the peptide

nitrogen[s] with the copper [II] nitrogen[s] with the copper [II] ions under alkaline conditions ions under alkaline conditions

reduction of reduction of Folin-Ciocalteu Folin-Ciocalteu reagentreagent, resulting in a color , resulting in a color change from yellow to blue, change from yellow to blue, which absorbs strongly at which absorbs strongly at 750nm 750nm

The Most Highly Cited Paper The Most Highly Cited Paper in Publishing History: Protein in Publishing History: Protein Determination by Oliver H. Determination by Oliver H. LowryLowry

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Advantages and disadvantages of Advantages and disadvantages of Lowry MethodLowry Method

More sensitive than the Biuret assay (can detect More sensitive than the Biuret assay (can detect lower concentrations of protein) lower concentrations of protein)

Absorption reaction is linearly dependent upon Absorption reaction is linearly dependent upon protein concentration, but only at low protein concentration, but only at low concentrations of protein (i.e. the standard curve concentrations of protein (i.e. the standard curve and assay must be performed at a low and assay must be performed at a low concentration regime). concentration regime).

More critical to timing and precision of person More critical to timing and precision of person doing the assay doing the assay

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Making a standard curve first Making a standard curve first with BSAwith BSA

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Today’s workToday’s work

Isolate the total protein:Isolate the total protein:

group 1 & 4: wt and pdi2 mutant group 1 & 4: wt and pdi2 mutant plantplant

group 2 & 3: wt and group 2 & 3: wt and gfp-2sc gfp-2sc plant plant

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GFPGFP GGreen reen FFluorescent luorescent

PProtein: a rotein: a fluorescent protein fluorescent protein isolated from isolated from jellyfish.jellyfish.

Its role is to Its role is to transduce the transduce the blueblue chemiluminescence chemiluminescence of the protein of the protein aequorinaequorin into green into green fluorescent light by fluorescent light by energy transferenergy transfer. .

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The use of GFP in researchThe use of GFP in research The gene for GFP has been The gene for GFP has been

isolatedisolated It has become a fluorescent It has become a fluorescent

protein tag to makeing protein tag to makeing chimeric proteins chimeric proteins

It has been expressed in It has been expressed in bacteria, yeast, slime mold, bacteria, yeast, slime mold, plants, drosophila, plants, drosophila, zebrafish, and in zebrafish, and in mammalian cells.mammalian cells.

PDIPDI GFPGFP

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GFP-2SC PlantGFP-2SC Plant

SP: the signal peptide of pumpkin 2S albumin

2SC: : Vacuolar-targeting signals of pumpkin 2S albumin

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ReferencesReferences http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlhttp://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. J. Biol.

Chem.Chem.193, 265–275193, 265–275 www.bio-itworld.comwww.bio-itworld.com/ archive/091103/russell.html/ archive/091103/russell.html http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmohttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo

cz/gfp.htmcz/gfp.htm