Updated: 2024-02-07T16:44:17
Created: 2024-02-07T16:37:48
Optional:
Inhibitors in 1 ml of distilled water, store at -20°C.
1 mM DTT
Phosphatase inhibitors cocktail (when phosphorylations need to be preserved; dilute 100x). Alternatively, make your own stocks of Na₃VO₄ (sodium orthovanadate). It can be used at a final concentration of 1 mM in your LB.
Na₃VO₄ Preparation:
For 20 ml of 10 mM Na₃VO₄, use 36.782 mg of Na₃VO₄.
Dissolve in double-distilled water.
Set the pH to 10 with HCl.
It could happen that the solution will turn yellow when adjusting the pH. Boil until it turns colorless.
Cool and adjust the pH to between 9-10. Repeat this cycle of boiling, cooling, and adjusting the pH until it stabilizes.
Bring up the volume.
Bryja Lab Protocol Series
Immunoprecipitation (IP) is a technique to isolate a protein antigen out of a solution using an antibody that specifically binds to that protein. This process can be used to:
Isolate and concentrate a specific protein from a sample containing many different proteins.
Analyze protein-protein interactions.
Detect and quantify the presence of known proteins.
Antibodies against all proteins must be available. If not, use tagged proteins and an overexpression system.
Usual amount of cells/reaction: 1x 10 cm plate.
Usual amount of protein: 200-500 µg/reaction.
It is preferred to measure the protein concentration in your lysates using the BCA assay and use 500 µg for IP.
This protocol describes the process for working with 10 cm plates and 6-well plates.
For 15 cm plates, scale up 3x.
Important: Keep the plates/samples on ice at all times once you begin the IP.
Calculate the amount of lysis buffer needed (1 ml/plate) + approx. 2 ml for equilibrating the beads.
To complete the lysis buffer, add:
1x protease inhibitors (get 50x protease inhibitors by dissolving 1 tablet of Roche #05-Immunoprecipitation to 20 ml or your desired volume). Store in aliquots at -20°C. Discard aliquots if they turn yellow.
0.01 M N-ethylmaleimid (NEM) to preserve ubiquitinations.
0.1% SDS to solubilize proteins bound to the cytoskeleton.
Suck up the medium from the plate.
Wash with 2 ml of PBS. Gently stir to wash the remaining medium, then suck up the PBS.
(Try to use cold PBS.)
Let the plates stand in a tilted position so that the remaining PBS collects at the bottom.
Suck up the PBS.
Note: The protocol can be interrupted at this stage, and "dry" cells on the plate can be frozen at -80°C.
Add 1 ml of lysis buffer and let the lysis continue for at least 15 minutes on ice. Gently swirl the plate occasionally to ensure the lysis buffer covers the plate uniformly.
Transfer the lysate to an Eppendorf tube and centrifuge at maximum speed at 4°C for at least 10 minutes (Max 20-25 minutes).
Divide the supernatant into new 1.5 ml Eppendorf tubes as per your experimental setup.
For standard procedure with two IP antibodies, transfer 400-450 µl of cleared lysate into each tube with the antibody.
Alternatively, transfer the supernatant into a fresh Eppendorf tube and measure the total protein concentrations. Once you have the values, divide your lysates, using 500 µg for your IP condition.
For TCL, add 60 µl of lysate to a 0.5 ml tube. Add 15 µl of 5x Laemmli buffer to your TCL samples. Store samples at -20°C until gel run.
Add 1 µg of antibody to each reaction.
(Check antibody concentrations on original vials or in datasheets.)
Example dilution: HA 1:150.
Incubate the antibody-lysate mixture for 30 minutes to an hour on ice.
(Modification: You may incubate overnight if the antibody affinity is low.)
Prepare G-protein sepharose beads (approx. 15 µl solid beads/30 µl slurry per reaction).
Equilibrate the G-protein sepharose beads by washing them twice in complete lysis buffer and centrifuging at 0.1 RCF, 4°C, for 1 minute.
Divide the beads into individual samples in a larger volume (approx. 40 µl/tube) to ensure even distribution of beads.
Place the micro test tubes into the carrousel in the fridge and let them rotate overnight (or at least for 4 hours at room temperature). (If using the modified step 10, incubate with the beads for 4 hours in the cold room, then proceed with washes.)
After incubation, take out the micro test tubes from the carousel, centrifuge at 0.1 RCF, 4°C, for 1 minute.
Carefully remove the supernatant and add 800 µl of lysis buffer without protease inhibitors to the beads.
Aim to mix the beads with the lysis buffer.
Centrifuge at 0.1 RCF, 4°C, for 1 minute.
Repeat steps 9 & 10 for 3-5 cycles.
Suck up the supernatant, then add 42 µl of 2x Laemmli buffer (this ensures two WB runs when 20 µl are loaded).
Boil all samples for 5 minutes and analyze by Western Blot (WB).
5 ml of 2M Tris pH 7.5 (final concentration 50 mM)
6 ml of 5M NaCl (final concentration 150 mM)
0.8 ml of 0.25M EDTA (final concentration 1 mM)
1 ml NP40 (final concentration 0.5%)
Make up the volume to 200 ml with distilled water.