Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

1. Immunoprecipitation using Protein A/G PLUS Agarose Beads

Cell Lysate Preparation

1. Centrifuge 10⁸ thymocytes in a microcentrifuge at 13,000 rpm for 3 min and remove the supernatant.
   Note: The cell number will vary depending on the expression levels of the desired protein and the chosen cell type.
2. Re-suspend the cells in 500 µL lysis buffer RIPA with PMSF.
3. Disrupt cells using a few quick pulses with a vortex and then aspirate the lysate a few times with a 25 G needle attached to a syringe.
   Note: Avoid creating bubbles. Use a larger needle such as a 21G needle for larger cell types.
4. Incubate the cell lysate on ice for 10 min.
5. Centrifuge the lysate at 13,000 rpm for 15 min at 4°C.
6. Transfer the supernatant to a fresh, labeled microcentrifuge tube.

Pre-clearing

7. Add 20 µL Protein A/G PLUS agarose beads and 1 µg of an isotype control antibody (here, mouse IgG1 isotype control antibody is used), to the lysate.
   Note: The choice of isotype antibody used will depend on the protein-specific antibody used in later in the pull-down step.
8. Incubate the lysate mix on a centrifuge rotator in the cold room (4°C) for 30 min.
9. Centrifuge the sample at 3200 rpm for 30 s at 4°C.
10. Transfer the pre-cleared supernatant to a fresh, labeled, 1.5 mL microcentrifuge tube. Discard the pellet.

Protein Concentration Determination

11. Determine the protein concentration of the cell lysate by performing a Bradford Assay.
12. Aliquot 1000 µL Bradford Reagent into 7 microcentrifuge tubes.
13. Add the following amounts of BSA protein standard (2 mg/mL) into 6 of the tubes (Table 1).

Tube Number	BSA Volume (μL) (2 mg/mL)	Protein Concentration (μg/μL)
1	0	0
2	1	2
3	2	4
4	3	6
5	4	8
6	5	10

Table 1: BSA protein standard amounts

14. In the 7^th tube, add 1 µL of the pre-cleared lysate.
    Note: To ensure that the sample concentration lies within the assay detection range, prepare and analyze a 1:2 or 1:5 lysate dilution as well.
15. Place 200 µL from each of the 7 tubes into individual wells of a flat-bottom, 96-well plate repeating each sample in triplicate.
16. Read plate on a plate reader at 595 nm.
17. Generate the standard curve in excel and calculate the protein concentration of the pre-cleared lysate.

Pull down

18. Label two fresh 1.5 mL microcentrifuge tubes- one as 'control' and other as 'test,' which in this example is c-myc.
19. Place 500 µg of pre-cleared lysate into each of these tubes.
    Note: The amount of protein used here will depend on the amount of the protein desired to be purified.
20. Bring up the total volume for each tube up to 500 µL using lysis buffer.
21. Add 2 µg of anti-c-myc antibody to the test group tube and 2 µg mouse IgG1 isotype control antibody to the control group.
    Note: Quantity of antibody will depend upon antibody efficacy and the amount of the target protein.
22. Incubate the tubes on a rotator in the cold room (4°C) for 2 h.
23. Add 20 µL Protein A/G PLUS agarose beads to each tube.
    Note: It is advisable to use pipette tips with the end cut off to prevent damage to the beads.
24. Incubate on a rotator in cold room (4°C) overnight.
    Note: Depending on the target protein and the antibody efficacy, this step can vary from 1 h to overnight.
25. Centrifuge the tubes at 3200 rpm for at 30 s 4°C to pull down the beads.
26. Aspirate the supernatant from each tube.
    Note: The target protein is now bound to the beads.
27. Wash the beads two times using 500 µL 1X Dulbecco's PBS.
28. Centrifuge the tubes at 3200 rpm for at 30 s 4°C.
    Note: For more stringent washing, use more stringent buffers, such as RIPA.
29. Aspirate the buffer from each tube. Using gel-loading tips, remove any leftover buffer from beads and keep the beads on ice to elute the protein.
    Note: In this example, the protein is eluted into SDS-PAGE running buffer by boiling the beads, for Western blot analysis. This approach is suitable for verifying IP results or to examine protein-protein interactions. For other downstream applications, such as purification of proteins for structural or enzymatic analysis, more sophisticated systems, such as epitope tags (flag-tag or myc-tag) are used to avoid elution of the antibody with the protein of interest.

2. IP Verification Through Western Blot Analysis

SDS-PAGE Electrophoresis:

1. Re-suspend beads in 20 µL SDS-PAGE loading dye containing β-mercapto-ethonol.
2. Boil the samples at 95 °C for 5 min.
3. Centrifuge the beads at 13,000 rpm for 10 s at room temperature.
4. Using gel loading tips, carefully pipet the samples obtained from the beads and load them into wells of 4-15% gradient SDS-PAGE gel.
5. In addition to the samples, load a lane with a protein ladder as well as a lane with the pre-cleared lysate to serve as a loading control.
6. Run at 100 V until dye front reaches the bottom of the gel (~1h).

Western Blot Analysis:

7. Make Western blot sandwich, ensuring that PVDF membrane is between gel and red cathode.
8. Transfer for 1 h at 100 V.
9. Place the membrane in 5 mL blocking buffer at room temperature for 1 h on a rocker at a low setting, to block the non-specific protein binding sites.
   Note: The volumes of blocking buffer, primary antibody, secondary antibody, and washes may need to be increased for larger sized blots.
10. Incubate the blot with 5 mL anti-c-myc antibody in the blocking buffer overnight at 4°C on rocker at a low setting.
    Note: The antibody used here should be different than the one used in the pull-down step.
11. Wash the blot 3-6 times using 5 mL TBST with each wash being 5 min at room temperature on a rocker at a low setting.
12. Incubate the blot with HRP-tagged anti-rabbit light chain secondary antibody in blocking buffer, for 1 h at room temperature on rocker at a low setting.
    Note: The choice of secondary antibody will depend on primary antibody used for the Western blot. Additionally, a light chain specific secondary is used in the protocol because the target protein is close in molecular weight to the heavy chain of the antibody. If the target protein is close to 50kDa, use a light chain specific secondary. If the target protein is close of 25kDa, use and heavy chain specific secondary.
13. Wash the blot 3-6 times using 5 mL TBST with each wash being 5 min at room temperature on a rocker at a low setting.
14. Remove liquid from blot and dab edge of blot on laboratory wipes to remove excess liquid.
15. Cover blot with 1x chemiluminescent detection reagent' and incubate for 1 min.
    Note: The following steps should be done in quick succession as the detection reagent is light and time sensitive.
16. Dab edge of blot on laboratory wipes to remove excess detection reagent.
17. Place the blot on imaging surface of Imager tray.
    Note: Chemiluminescent blots can also be visualized using film.
18. Image using the 'Chemiluminescent Program' to capture multiple time points from 10 s to 5 min.
    Note: The optimal time may change based on protein quantity and the quality of antibody.
19. Chose an image with optimal band visibility and then export that image.
20. Prior to moving the blot, take a picture of the blot using the imager, to capture the location of the ladder. Then, export that image as well.
21. Using a slide preparation software (such as PowerPoint), align the bands and ladder images to form a single image.