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Immunoprecipitation

Described here is an Immunoprecipitation protocol, a procedure used to identify and bind target proteins in solution; These proteins are then available for further characterization by SDS polyacrylamide gel electrophoresis, Western blots or other techniques. For example, your primary Chicken IgY could be mixed with a complex mixture of antigens in solution (often a cell lysate). The antigen-antibody complexes that form can be immunoprecipitated using a solid phase reagent that would bind to the immune complexes. When your primary antibody is a mammalian IgG, the reagent is often Protein A or G coupled to an agarose bead that is used as the immunoprecipitating reagent. IgY does not bind to Protein A or Protein G, so a secondary (anti-IgY) linked to agarose acts as the immunoprecipitating reagent when Chicken IgY is the primary antibody.

Immunoprecipitation

Reagents and Buffers:

50 mM Tris Buffer, pH 8.0

Dissolve 0.6 grams Tris Base in 50 ml distilled water. Adjust the pH to 8.0 with concentrated HCl.

QC to volume of 100 ml. Store in fridge.

Lysis Buffer (NP-40) (50 mM Tris, 1% NP-40, 15 mM NaCl):

To 25 ml of 50 mM Tris buffer, pH 8.0 (see above), add:
0.44 gm NaCl
1.0 ml NP-40

QC to volume of 50 ml with 50 mM Tris buffer, pH 8.0 and mix thoroughly. Store in refrigerator.

Lysis Buffer (RIPA) (50 mM Tris, 1% NP-40, 15 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate):

To 25 ml of 50 mM Tris buffer, pH 8.0 (see above), add:
0.44 gm NaCl
1.0 ml NP-40
0.05 gm SDS
0.25 gm Sodium deoxycholate

QC to volume of 50 ml with 50 mM Tris buffer, pH 8.0 and mix thoroughly. Store in refrigerator.

1 M Potassium Phosphate Buffer, pH 7.3

660 ml 1 M K2HPO4
330 ml 1 M KH2PO4
Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH2PO4; to raise pH, add more 1M K2HPO4). Store in fridge.

Phosphate Buffered Saline (PBS)

30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3

QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.

Phosphate Buffered Saline-Tween, 0.25% (PBS-Tween)

Add 2.5 mL Tween 20 to 1 L PBS.

Procedure

The preparation of your cell lysate will depend on the type of cell used – whether the cell is prokaryotic or eukaryotic, has a cell wall, whether the antigen must retain tertiary structure or biological activity, for example will determine how you prepare your lysate. There are many recipes and experimentation may be required. Using Antibodies, A Laboratory Manual, addresses in detail various protocols. Here, suffice to say that there are 2 common cell lysis buffers: NP-40 and RIPA (see above). Triton X-100 can be substituted for the NP-40 if desired. The former is a less denaturing buffer and might be tried initially to determine if it effectively releases the desired antigen. A variety of protease inhibitors may be added to the lysis buffers to prevent enzyme activity.

Making your Cell Lysate:

  1. Wash cells once with room temperature PBS. Prepare your cell lysate in the cold room, using cold buffers as proteolytic denaturation occurs quickly once the cells lyse. For cultured cells, add cold lysis buffer to the washed cell pellet and gently shake on shaker. Instead of placing on a shaker, bacterial cells require sonication: 4 pulses of between 10-30 seconds should suffice. Recommended for yeast cells is mechanical shearing of the cells: Add an equal volume of glass beads (prewashed twice in 1 N hydrochloric acid and lysis buffer), to your yeast cells suspended in lysis buffer. Vortex vigourously in 30 second pulses until cells are lysed.
  2. Spin for 10 minutes at 10,000g in the cold. Remove the supernatant, careful to not disturb the cell pellet. Supernatants should be kept cold.

Preclearing your Lysate

Preclearing is a technique used to optimize the immunoprecipitation reaction by clearing the supernatant of proteins that may non-specifically bind to immune complexes, increasing the background of your reaction. It is done by mimicking the immunoprecipitation procedure but using a pre-immune or non-specific irrelevant antibody from the primary antibody producing animal. In some immunoprecipitation reactions, this step may not be necessary.

  1. Add Normal Chicken IgY (approximately 0.01 x lysate volume) to your cell lysate and incubate for 1 hour in the cold.
  2. Now add 0.1 x lysate volume of packed (or 0.2 ml resuspended agarose) Donkey anti-IgY agarose to the mixture and incubate for another 30 minutes in the cold.
  3. Centrifuge mixture at 10,000g for 15 min in the cold, and then carefully collect the supernatant. Now your sample is ready for your Primary Chicken IgY antibody.

Preparing your Immune Complexes

The most efficient immunoprecipitation reaction is one that uses an affinity purified IgY. This is because all of the IgY present will be antigen-specific and therefore most or all of the immune complexes precipitated will contain antigen. Using a total IgY preparation will result in only between 0.5 and 5% of the precipitated immune complexes containing antigen.

  1. In the cold room (or on ice), add your lysate aliquot into your 1.5 ml microtubes. Bring the volume up to 0.5 ml with lysis buffer. Add between 0.5 and 5 ug of your primary IgY antibody per 100 ug cell lysate. Incubate in the cold for 1 hour.
  2. Add 200 ul (50% slurry) Donkey anti-IgY agarose (20% vol/vol in lysis buffer) to each tube. Incubate again for 1 hour in the cold, rocking.
  3. Centrifuge the agarose bead immunoprecipitates at 10,000 x g for 15 seconds. Discard supernatant by aspiration and wash agarose pellet 3 times with lysis buffer.
  4. After the final wash and complete aspiration, your antigen-containing immune complexes are available for further analysis.
  5. If you want to free the antigen from the immune complexed agarose, you may want to add elution buffer to the complexes (please see Affinity Purification of IgY).

References

Ed Harlow & David Lane, Eds. Using Antibodies : A Laboratory Manual. Chapter 7. Cold Spring Harbor Laboratory Press, New York, 1999.

Dai, L, Taylor, MS, O'Donnell, KA and JD Boeke. 2012. Poly (A) Binding Protein C1 is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation. Mol Cell Biol. 32:4323-4336.

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