In vitro pull down and Co-IP assays

In vitro pull down assay

Binding/Washing Buffer

* pH of the stock solution at room temperature. This is subject to change depending on the isoelectric point of your protein of interest. Try to keep a difference of at least one pH unit. Also, be aware that the pH of Tris buffers changes with temperature. This protocol is performed at 4°C, so the actual pH is ~ 0.5°C higher during the experiment.

** Add right before use.

Protocol

1. In a 2 ml tube mix:
   1. 2.5 ug prey protein times the number of samples (in my case, GST-fusion).
   2. 50 ul (bed volume) of bait resin (in my case, amylose resin)
   3. 1-2 ml of Binding buffer
2. Pre-absorb the prey proteins by gently rotating 1h in 4°C
   1. Prepare the tubes with the bait proteins ON ICE:
      1. 1 ml Binding Buffer
      2. 2.5 ug Bait protein (in my case, MBP-fusion)
3. Clear the proteins by centrifugation for 2 minutes at 13,800 rcf in 4°C.
4. Transfer 2.5 ug of prey protein to each bait protein tube. Specifically, divide the volume of clarified protein in the total number of samples and split evenly.
5. Incubate rotating for 2 hours in room temperature.
   1. Take aside the bait resin (in my case, amylose resin) that you will use fir the experiment. I use 50 ul (bed volume) of bait resin per sample.
   2. Equilibrate the bait resin with Binding Buffer. I do this by washing it with binding buffer 3 times. To collect the resin without damaging it, I centrifuge at < 600 rcf.
   3. Make protein gels for:
      1. Input control
      2. Western blot for pray proteins
      3. Coomassie staining for bait proteins
6. Add the 50 ul (bed volume) bait resin, and continue incubation in the same conditions for 2 more hours in room temperature.
7. Wash 3 times by adding 1ml of the binding buffer and rotating 10 minutes, collect the resin by centrifugation for 2 minutes at 1,500 rcf.
8. Wash 3 more times with the binding buffer without the 10 minutes rotation intervals. The last time, take all the liquid out.
9. Add 50 ul of 2X SDS and resuspend the beads. Boil 5 minutes at 95°C, follow by 2 minutes on ice, and 2 minutes of centrifugation at > 13,000 rcf in 4°C.
10. Load 10ul in gels.

Immunoprecipitation from Nicotiana benthamiana agro-infiltrated leaves

Buffers

* pH of the stock solution at room temperature. This is subject to change depending on the isoelectric point of your protein of interest. Try to keep a difference of at least one pH unit. Also, be aware that the pH of Tris buffers changes with temperature. This protocol is performed at 4°C, so the actual pH is ~ 0.5°C higher during the experiment.

** Add right before use.

Protocol

1. Grind 0.4 g of each sample in liquid Nitrogen using mortar and pestle.
2. Mix the powder with 1.2 ml IP buffer (3 ml buffer : 1 g sample).
3. Thaw the samples by gentle continuous stirring in the mortar, do not get bubbles. This step shall be done on ice. Transfer the liquid to a 2 ml tube.
4. Centrifuge 15 minutes at full speed in 4°C.
   1. While the sample is clarified, prepare 1.5 ml centrifugation tubes by cooling them on ice.
   2. Also, take the anti-Myc beads and keep them on ice.
5. Take out the supernatant to the cold 1.5 centrifugation tubes and centrifuge again for 15 minutes at maximum speed (21,000 g) in 4°C.
   1. Prepare The anti-Myc-Conjugated Agarose Beads:
      1. Prepare 15 ul of beads per sample to be assayed.
      2. Spin down for 3 minutes at 1,600 g in 4°C. Remove the supernatant.
      3. Wash 3 times with the IP buffer; each time add ±1 ml of IP buffer and invert several times until the beads are completely in solution, then centrifuge 3 minute at 1,600g in 4°C. Remove the liquid.
      4. The last time, remove the liquid and keep the beads on ice.
6. Collect the input sample by putting 50 ul of the supernatant from step 5 to a clean tube, then add 10 ul of 6X SDS buffer.
7. Take the remaining supernatant (clarified solubilized protein extract) out to incubation with the anti-Myc conjugated beads from step 5.a.iv. Add the supernatant to the tubes with the equilibrated beads and incubate rotating for 2h in 4°C.
8. After the incubation, take the tubes back and place them on ice for about 5 min to decant the agarose beads, then spin down at 1,600 g for 3min.
9. Wash the beads 5 times as follows: add 1 ml of IP buffer, rotate in 4°C for 10 minutes, collect the beads by centrifugation for 2 minutes at 1,600 g in 4°C. 
10. Do a sixth wash without rotation 
11. Elute with 400 µL of the Elution buffer; rotate in room temperature for 20 min then centrifuge at 1,600g for 3 min and transfer the supernatant to a clean tube (basic elution samples); store at -80℃.
12. Regeneration of anti-Myc-conjugated agarose beads:
    1. After elution, immediately wash the anti-Myc agarose beads with the IP buffer for 3 times before storing the beads in 50% suspension with glycerol-TBS-NaN₃ (50 mM Tris HCl pH 7.5, 150 mM NaCl, 50% Glycerol, 0.02% NaN₃) buffer at 4°C. All the beads can be combined.

Evaluation

1. Run the gels for western blot. For IP, probe with anti-Myc antibody; for CoIP, probe with the antibody that is pertinent to detect the co-immunoprecipitated protein. 
   1. 10 ul of each basic elution sample is diluted with 10 ul of 2X SDS buffer, mixed, boiled at 95°C for 5 minutes and spun down. 
   2. Use 2 ul of each sample to run the gels.

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