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Laboratory Activity Six

ProteinsExtraction, Precipitation &

Quantification

Purpose of Lab 06

Introduction to the theory, concerns & applications in the handling of proteins for biochemical studies.

Specifically:

Tissue disruption & protein extraction.

Salting in vs. salting out & ammonium sulfate fractionation.

Treatments that promote protein denaturation

Treatments that promote protein “protection”.

Methods for protein quantification.

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Underlying Premises of Protein Handling

Proteins are most stable & functional in their native environment (i.e. the cells & tissues in which they are found). pH = 7.4 Osmolality = 0.3 – 0.4 Compartmentalized. Reducing environment.

Many biochemical studies involve removal/purification of proteins from their cellular environments.

Overall concern is to keep isolated proteins in their most functional condition during handling and studies.

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Important Concerns When Studying & Handling Proteins: Choice of tissue disruption method to release protein(s) of

interest.

Protection of protein extracts & components from endogenous & exogenous factors (i.e. maintenance of protein integrity).

Concentration & purification of individual protein components.

Estimation of protein concentrations / amounts.

Diversity of protein structures & functions.

Introduction to Protein Handling

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Common Tissue Disruption Methods

Main Concerns: Sheer forces vs. tissue/cell disruption vs. organelle &

protein damage/destruction. Sample dilution into homogenization buffer; thermal

denaturation. Release of toxic cellular contents; exposure to oxidizing

conditions. 5

Toxic Cellular Components ?

Hydrolytic enzymes of lysosomes & plant vacuoles.

Phenolics, pigments, acids, ions of vacuoles.

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Polyvynylpolypyrrolidone(PVPP)

Tannic Acid

Common Additives to Tissue Homogenization Media

Buffers Thiol Reagents Protease Inhibitors Osmoticants Detergents PVPP Low (non-freezing)

Temperatures Others (lab manual)

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Collectively referred to as the homogenization buffer or the homogenization “cocktail”.

Most Homogenizations Greatly Dilute Out Cellular Contents

Cell-Free Extracts Often Require “Reconcentration”: Lyophilization (freeze-drying). Reverse Osmosis.

Salting Out. Centrifugation (lab 10).

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Semi-PermeableMembrane

AddedPressure

PureSolvent

Solute ofInterest

Small-scale Lyophilizer

Salting In & Out of Proteins

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So

lub

ilit

y

Salt Concentration

Salting in

Salting out

ProteinMolecules

Cations: N(CH3)3

+ > NH4+ > K+ > Li+ > Mg2+ > Ca2+ > Al3+ > guanidinium

Anions: SO4

2- > HPO42- > CH3COO- > citrate > tartrate > F- > Cl- > Br- > I- > NO3

- > ClO4-

> SCN-

Salting In & Out of Proteins

Some Important Notes:

NH4SO4 is normally the salt of choice. Relatively high solubility ( 4.0M; 528 g/L; 52.8% w/v). Concentrated solutions have low densities; do not interfere with

protein sedimentation. Concentrated solutions are anti-microbial. Concentrated solutions protect most proteins against

denaturation.

Different proteins have different solubility curves. Differential salting out can be used for purification.

Salt concentrations are expressed as a % of saturation. % Saturation ≠ % (w/w) or % (w/v). 100% saturation = 4.1M @ 20C; 3.9M@ 0C.

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Activity 1: Preparation of a Cell-Free Leaf Extract (TA’s)

Preparation of a Cell-free Extract from Spinach Leaves:

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1 2 3 4 5 6

Activity 2: Students test the effects of different treatments on protein denaturation.

Protein Denaturation

Defined as . . .The disruption (or unfolding) of tertiary & secondary protein structure that leads to loss of protein function.

Often manifested as the formation of cloudy precipitates or flocculation of protein.

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Activity 3: NH4SO4 Fractionation of a Cell-Free Leaf Extract (TA’s)

NH4SO4 Fractionation of Spinach Cell-free Extract:

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• Add (NH4)2SO4 to 50% saturation.(31.3g per 100 mL of extract)

• Equilibrate for 30 min.• Centrifuge @10,000 x g for 10 min.

50%Supt.

• Redissolve in fresh extraction buffer.

Bradford / Coomassie Blue Protein Assay

50%Pellet

Cell-Free Leaf Extract

TA’s

UV-280Protein Assay

+Students(Activities 4 & 5)

Save fractions for electrophoresis (in two weeks).• Recovery or

purification of Rubisco subunits.

383 mL

450 mL

27 mL

Activity 4: UV-280 Protein Determination

Based on absorbance of aromatic amino acids @ 280 nm.

[Protein], mg/mL = ABS280 x 1.55

[280 0.645 mL/(mgcm)].

Advantages: Simple, non-destructive. Fairly sensitive (20µg/mL - 3

mg/mL).

Disadvantages: Contaminants (especially nucleic

acids) also absorb @ 280 nm. Variable amounts of aromatic

amino acids in various proteins.

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H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

OH

H2N CH C

CH2

OH

O

HN

H2N CH C

CH2

OH

O

N

NH

Phe Tyr

Trp His

Activity 5: Bradford Protein Assay

Based on the quantitative colorimetric shift that occurs when “Coomassie Brilliant Blue G-250” binds with proteins.

Binding promoted by hydrophobic & electrostatic interactions.

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+

H

H

+

-

-

H+

Basic Amino Acids(in protein structure)

Coomassie Brilliant Blue G-250 Colorimetric Reactions

Anionic form bound to protein(max = 610 nm)

Cationic form(max = 470 nm)

Bradford Protein Assay

Advantages: Simple, quick, sensitive assay (1 – 200

µg/mL). Few interfering substances.

Disadvantages: Unstable color reaction. Relatively high blanks ( 0.400A). Not perfectly linear (i.e. limited linearity). Different proteins bind different amounts

of dye.

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Sample Bradford Data

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Bradford Estimates of 10 mg/mL

SolutionsProtein Conc.*Pepsin 4.1-Globulin (rabbit)8.0Lysozyme 9.9Histones 15.8BSA 21.1Cytochrome c 25.3(*Data from Appendix XII;

standard was Bovine -Globulin)

The Biuret Reaction*

A quantitative test for protein (1 – 10 mg/mL). Based on a colorimetric shift from blue to purple (max = 540 nm). Involves the formation of a “tetra-dentate” Cu2+-protein complex.

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Protein + Biuret Reagent

NaOH, CuSO4 &Na/K-Tartrate

(Tetra-dentate Complex)

(*Performed as part of Activity 2)

Summary of Activities

TA’s prepare cell-free extract & NH4SO4 cuts. Students:

Test effects of various treatments on protein denaturation.

Quantify protein in various fractions via UV-280. Quantify protein in various fractions via Bradford.

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Questions or Comments

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