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Keywords:

  • Protein isolation;
  • immunoglobulin Y;
  • IgY;
  • chicken antibodies

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. QUESTIONS TO PROBE STUDENT UNDERSTANDING
  6. Acknowledgements
  7. REFERENCES

Separating individual proteins from complex mixtures of molecules is the basis of many biochemical investigations. The method describes the separation of immunoglobulin Y (IgY) from chicken eggs using a series of physical and chemical separation techniques. The separation is rapid, and the success of each step is readily viewed on an SDS-polyacrylamide gel. IgY identity can be confirmed on a Western blot probed with enzyme-labeled anti-IgY antibodies. The method is a good illustration of protein separation when there is no enzyme activity to follow.

Egg yolk provides the growing chick with nutrients and maternal antibodies. Egg yolk antibodies (immunoglobulin Y) are easily isolated using polyethylene glycol (PEG)11 precipitation. Chicken eggs are a preferable source of antibodies as the antibodies are found in concentrations comparable to the concentrations in chicken blood, and isolating the antibodies does not require venipuncture. Polson [1] introduced the use of the neutral water-soluble high molecular mass polymer (PEG) for the fractional precipitation of proteins. PEG is a mild precipitating agent that operates a steric exclusion mechanism concentrating proteins until they exceed their solubility limit [13].

We present a short protein isolation protocol to separate immunoglobulins from eggs. The steps during protein isolation are monitored by separating the proteins on a polyacrylamide electrophoresis gel run in the presence of sodium dodecyl sulfate and a reducing agent. The stained gel shows that at each step the number of proteins in the mixture is decreasing, and two major protein bands, representing the reduced heavy and light chains of IgY, are separated from a complex mixture of proteins. The identity of chicken immunoglobulins can be confirmed with enzyme-labeled rabbit anti-chicken antibodies on a Western blot of the polyacrylamide electrophoresis gel. The IgY isolation technique has been used successfully in a Biochemistry specialist course (2nd-year course) and in a Biochemistry techniques course for non-specialist students in their 3rd year of study.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. QUESTIONS TO PROBE STUDENT UNDERSTANDING
  6. Acknowledgements
  7. REFERENCES

IgY is separated from egg yolk using Polson's method [4, 5] with minor modifications. All reagents used are of analytical grade. The equipment required for this experiment is common to a biochemistry laboratory: magnetic stirrer (optional), centrifuge capable of 12,000 × g, UV spectrophotometer, two-place balance, pH meter, and polyacrylamide gel electrophoresis and electroblotting equipment. A single egg is sufficient for each student.

Separate yolks from the egg white. Excess white can be removed by carefully washing the egg yolk under running tap water. Puncture the yolk sac and determine the yolk volume in a measuring cylinder. Note the volume. The yolk sac is discarded. Two volumes of 100 mM phosphate buffer, pH 7.6 containing 0.02% (w/v) sodium azide are added to the yolk and mixed thoroughly. A glass rod can be used. The first sample (number 1, 50 μl) is taken at this stage. Add 3.5% (w/v) polyethylene glycol (PEG 6000 available from Merck, www.merck.de) and mix until the PEG has completely dissolved. The sample is centrifuged (4,420 × g for 20 min at room temperature). A cotton wool (absorbent-type) plug is firmly placed at the base of a funnel, and the supernatant is filtered through. The lipid fraction is trapped by the cotton wool. Note: the filtrate must be a clear yellow liquid and NOT milky in appearance; if the filtrate is not clear, it is recommended that the filtration step is repeated. The filtrate volume is recorded, sample number 2 (50 μl) is removed, and the PEG concentration is increased (it is already at 3.5%) to 12% (i.e. add 8.5%). Mix to completely dissolve the PEG and centrifuge (12,000 × g for 10 min at room temperature) Sample number 3 (50 μl) is taken from the supernatant, and the remaining supernatant is discarded. The pellet is dissolved in a volume of phosphate buffer equal to the egg yolk volume (see above). Add PEG to a final concentration of 12% (w/v), mix thoroughly, and centrifuge (12,000 × g for 10 min at room temperature). Take a sample (number 4, 50 μl) from the supernatant before discarding and resuspend the final pellet in ⅙ of the original egg yolk volume in phosphate buffer and keep a sample (number 5).

To determine of IgY concentration, read the A280 of a 1:50 dilution of the IgY in 100 mM phosphate buffer and calculate the concentration of IgY in the undiluted solution (extinction coefficient of IgY is E280 nm1 mg/ml = 1.25). For electrophoresis, 2 μl of samples 1, 2, 3, and 5 were added to 18 μl of phosphate buffer, and for sample 4, 20 μl of sample was used without additional buffer. An equal volume (20 μl) of Laemmli sample buffer [6] containing reducing agent (2-mercaptoethanol) and bromphenol blue were added to all the samples. After boiling (90 s) 5 μl of each sample were added to a lane on a 12.5% Laemmli [6] SDS-polyacrylamide electrophoresis gel. The sample order is given in the legend of Fig. 1. We used the Amersham Biosciences molecular mass markers, which contained phosphorylase b, 97 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; and trypsin inhibitor, 20.1 kDa. Separated proteins on an identical polyacrylamide electrophoresis gel were transferred by Western transfer [7] to a nitrocellulose membrane, blocked with 5% (w/v) fat-free powdered milk in 100 mM phosphate buffer, pH 7.6 for 1 h at room temperature, washed in the buffer, and probed with a 1:12,000 dilution of horseradish peroxidase-conjugated rabbit anti-IgY heavy and light chain antibody (Sigma, www.sigma-aldrich.com) for 1 h at room temperature, and enzyme activity was detected with 4-chloro-1-naphthol in the presence of hydrogen peroxide.

Acrylamide is a potential neurotoxin, and azide is potentially explosive and toxic. Materials safety data sheets (MSDS) are available from www.sigmaaldrich.com. Polyethylene glycol is non-hazardous.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. QUESTIONS TO PROBE STUDENT UNDERSTANDING
  6. Acknowledgements
  7. REFERENCES

Each step in the protocol illustrates the principles used to separate a protein from other biological molecules and isolate a protein from a mixture of proteins without following enzyme activity. The initial step is a physical method, and the proteins avidin, lysozyme, and a portion of the total egg ovalbumin, found in the egg white, are separated from the yolk contents by removing the egg white. Lipoproteins in the yolk include the yellow-colored vitellin fraction, and these are removed in the second step where they form a lipid-rich, insoluble “paste-like” mass. The remaining proteins are known as the levitins and are composed of ovalbumin (α levitin), α-2 glycoprotein (β levitin), and IgY (immunoglobulin or γ levitin) [4]. When the polyethylene glycol concentration in the filtrate is increased to 12% the solution becomes “milky” as the protein precipitates. This is a step where mistakes are often made by some students who do not realize that the filtrate already contains 3.5% PEG. When insufficient PEG is added the solution does not go milky. We routinely obtain a final pellet with a 90% purity of IgY, and yields are in the order of 60 mg of isolated protein from each egg.

Fig. 1 illustrates a complex mixture of proteins in lane 2 and the isolated protein comprising two protein bands in lane 6. A comparison between lanes 2 and 3 shows that many of the proteins present in the yolk (lane 2) are removed in the first PEG precipitation step (lane 3). We chose the ovalbumin band at 45 kDa as an example of a protein to monitor as the protein is readily seen as a band in lanes 2, 3, and 4 indicating that the protein is present in egg yolk, the filtrate, and then the first supernatant. A small quantity of the protein, seen as a faint band, is found in the final pellet. We thus chose molecular mass standards containing ovalbumin to aid in interpretation of Fig. 1. The bands at 67 and 27 kDa, representing the heavy and light chains of IgY, are faint in lane 2 and indicate the low concentration of IgY relative to other proteins in the egg yolk. Lanes 4 and 5 are included as they show that proteins that do not appear in the final pellet are left in each supernatant and illustrate the need to repeat the precipitation step to remove the few remaining contaminating proteins. The sample in lane 5 contained a 10-fold increase in sample volume to run sufficient proteins in the lane to be visualized with staining. The final pellet contains two major bands (lane 6) representing the heavy and light chains of IgY, respectively. It should be remembered that the isolated IgY represents a collection of antibodies against multiple antigens, and therefore the protein band on the gel may vary in width. IgY has a molecular mass of 180 kDa [8]. We have found that the IgY light chain from some egg yolks may stain lightly with Coomassie Blue. Using the extinction coefficient for IgY the yield in milligrams of protein can be calculated [9].

Fig. 2 illustrates the result of probing a Western blot of a gel identical to that presented in Fig. 1 with anti-IgY antibodies. The anti-IgY antibodies detect the heavy and light chains of the immunoglobulin in each of the steps of the isolation protocol. The final protein pellet appears to have two major and some minor bands present (Fig. 1, lane 6). The anti-IgY antibody confirms that, although there appear to be contaminating proteins present, the majority of the protein bands on the gel are IgY (Fig. 2, lane 6). Occasionally the IgY is incompletely reduced, and this can result in several other bands of IgY. The Western blot further illustrates that the isolated protein is mostly IgY.

Polyethylene glycol is an excellent method to precipitate a specific protein from a complex mixture of proteins. Polyethylene glycol has been used to isolate IgG from the sera of a number of animals [10], acetyl-CoA carboxylase from lactating rat mammary gland [11], and IgY in this procedure. IgY can be isolated from eggs using a water dilution method [12], ammonium sulfate [13], dextran sulfate [14], and ion exchange chromatography [15]. The technique described in this experiment with polyethylene glycol is widely accepted as the standard technique to isolate IgY [10]. Typically IgY of 89% purity is obtained compared with 70–80% with dextran sulfate or ion exchange chromatography or 60–70% with ammonium sulfate [10]. The polyethylene glycol protocol does not require dialysis steps and therefore can be completed in under 3 h.

There are several advantages to this procedure. Eggs are cheap and readily available, antibody levels in yolks are high, and one can avoid bleeding and other invasive techniques normally required to obtain antibodies from animals. The method is simple, there are few steps, and yields are high. We use the method to illustrate the isolation of proteins without access to a marker, such as enzyme activity. We have added a second experiment whereby IgY can be identified with an appropriate antibody on a Western blot. We find the protocol to be an excellent experimental teaching tool as the separation achieved at each step is readily observed on the gel. The separation can be run in an afternoon, and the samples can be stored until it is convenient to run them on a polyacrylamide gel. When adding the Western blotting step a further 3-h practical session is required. The isolated IgY should not be frozen and can be stored at 4 °C for several years. The isolated IgY can be used as source material for protein and immunological studies. IgY isolated in this manner has been used to study plant viruses [4], enzymes [16], and parasite proteases [17].

QUESTIONS TO PROBE STUDENT UNDERSTANDING

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. QUESTIONS TO PROBE STUDENT UNDERSTANDING
  6. Acknowledgements
  7. REFERENCES
  • Describe the physical and chemical techniques used at each step to separate proteins, lipids, and IgY from egg yolk.

  • Explain why there are two major bands present in the final product.

  • Design an experiment to show that the isolated IgY consists of a pool of polyclonal antibodies.

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Figure Figure 1. Proteins from each step in the isolation of immunoglobulin (IgY) from egg yolk analyzed on a 12.5% SDS-polyacrylamide gel run in the presence of 2-mercaptoethanol.Lanes 1 and 7, Amersham Biosciences molecular mass markers: phosphorylase b, 97 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa. Lane 2, buffered yolk (sample number 1); lane 3, filtrate (sample number 2); lane 4, second supernatant (sample number 3); lane 5, third supernatant (sample number 4); lane 6, final pellet of isolated IgY (sample number 5).

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thumbnail image

Figure Figure 2. Proteins from each step in the isolation of immunoglobulin (IgY) from egg yolk fromFig. 1. transferred to nitrocellulose and probed with rabbit anti-chicken antibody.Lane 1, buffered yolk (sample number 1); lane 2, filtrate (sample number 2); lane 3, second supernatant (sample number 3); lane 4, third supernatant (sample number 4); lane 5, final pellet of isolated IgY (sample number 5).

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  • 1

    The abbreviation used is: PEG, polyethylene glycol.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. QUESTIONS TO PROBE STUDENT UNDERSTANDING
  6. Acknowledgements
  7. REFERENCES
  • 1
    A. Polson, G. M. Potgieter, J. F. Largier, G. E. F. Mears, F. J. Joubert (1964) The fractionation of protein mixtures by linear polymers of high molecular weight, Biochim. Biophys. Acta 82, 463475.
  • 2
    R. F. Boyer (1993) Modern Experimental Biochemistry, 2nd ed., Benjamin/Cummings, San Francisco, CA, pp. 249250.
  • 3
    K. C. Ingham, in M. P.Deutscher, Ed. (1990) Methods in Enzymology, Vol. 182, Academic Press, San Diego, CA, pp. 301306.
  • 4
    A. Polson, M. B. von Wechmar, M. H. V. van Regenmortel (1980) Isolation of viral IgY antibodies from yolks of immunized hens, Immunol. Commun. 9, 475493.
  • 5
    A. Polson, T. Coetzer, J. Kruger, E. von Maltzahn, K. J. van der Merwe (1985) Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens, Immunol. Investig. 14, 323327.
  • 6
    U. K. Laemmli (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680685.
  • 7
    H. Towbin, T. Staehelin, J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A. 76, 43504354.
  • 8
    G. W. Warr, K. E. Magor, D. A. Higgins (1995) IgY: clues to the origin of modern antibodies, Immunol. Today 16, 392398.
  • 9
    T. H. T. Coetzer (1993) Type IV Collagenase and Cathepsins L and H: Proteinases Involved in Tumour Invasion. Ph.D. thesis, University of Natal, Pietermaritzburg, South Africa.
  • 10
    R.Schade, I.Nehn, M.Erhard, A.Hlinak, C.Staak, Eds. (2001) Chicken Egg Yolk Antibodies, Production and Application. Springer-Verlag, Berlin, pp 65107.
  • 11
    D. G. Hardie, P. S. Guy, P. Cohen, in J.Lowenstein, Ed. (1981) Methods in Enzymology, Vol. 71, Academic Press, San Diego, CA, pp. 2633.
  • 12
    E. M. Akita, S. Nakai (1993) Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with enterogenic E. coli strain, J. Immunol. Methods 160, 207214.
  • 13
    J. Wallman, C. Staak, E. Luge (1990) Einfache methode zur isolierung von immunoglobulin (Y) aus eiern immunisierter Hühner, J. Vet. Med. B37, 317320.
  • 14
    J. C. Jensenius, I. Anderson, J. Hau, M. Crone, C. Koch (1981) Eggs: conveniently packaged antibodies, methods for purification of yolk IgG, J. Immunol. Methods 46, 6368.
  • 15
    R. Schade, A. Schniering, A. Hlinak (1992) Die Gewinnung spezifischer polyklonaler Antikörper aus dem dotter von Hühnereiern als alternative zur immunisierung von Säugern-eine Übersicht, ALTEX 9, 4356.
  • 16
    T. H. T. Coetzer, R. N. Pike, C. Dennison (1992) Localization of an immunoinhibitory epitope of the cysteine proteinase, cathepsin L, Immunol. Investig. 21, 495506.
  • 17
    L. Troeberg, R. N. Pike, J. D. Lonsdale-Eccles, T. H. T. Coetzer (1997) Production of anti-peptide antibodies against trypanopain-Tb from Trypanosoma brucei brucei: effects of antibodies on enzyme activity against Z-Phe-Arg-AMC, Immunopharmacology 36, 295303.