Andrew L. Smith, Reynolds Lab; Proteomics Support Network

September, 2010

Note: This protocol has recently been accepted for publication in PLoS One. Please cite it appropriately.

Considerations before and during an IP-MS Project

Antibody

  • Does your antibody IP efficiently?  If not, then bead conjugation won’t help.
  • Is your antibody specific?  If you routinely see lots of extra bands on westerns, you will be IPing those proteins too. This will result in a lot of noise and background in your final MS results
  • What is the status of your antibody (i.e. purified IgG, sera, ascites)?
  • What you really want is purified IgG. When dealing with sera and ascites, you really don’t know how much antibody you have, and what really is binding to your beads during conjugation. Components within sera and ascites may also compete with your IgG for binding, or interfere with IgG conjugation and subsequent crosslinking. When mass spec is your goal, it is worth your effort to affinity purify your antibody prior to conjugating beads.
  • Will this bead conjugation affect your antibodies IP efficiency?
  • In some cases, crosslinking with DMP will modify the antibody and prevent it from IPing properly. It is absolutely necessary to test IP efficiency of your conjugated beads before scaling up.

Lysis and Immunoprecipitation

  • Is there a specific condition that promotes your complex of interest?
  • Growth condition? Stimulation? Lysis condition?
  • Do you know how much material you need? How abundant is your target protein?
  • Low abundance = more starting material necessary
  • Can you successfully co-IP with your antibody, cells, and lysis conditions?
  • Looking for the co-IP of a known interactor is the best way to determine if you antibody, lysis, and IP conditions will work.
  • Do you have a good negative control?
  • Irrelevant IgG, knockdown/knockout cell line, etc.
  • Have you optimized at small scale first?
  • Determine the volume of beads you will need for a robust IP
  • Determine your optimal elution condition

This all boils down to one thing: confidence in your sample. If you have properly optimized, you can be confident in the quality of your sample. This makes analyzing your complex mass spec data much, much easier.

Preparing Beads

Target protein is immunoprecipitated from whole cell lysates using an antibody covalently bound (via cross-linker) to Protein G magnetic beads (Dynabeads, Invitrogen). These beads have less background than sepharose and magnetic separation allows for quicker and gentler washes.

Keep in mind these conditions are guidelines, not gospel. In some cases, different binding buffers, crosslinkers, etc may work just as well, or better.

I routinely prepare 0.5-1mL of dynabeads at a time. 1 mL of slurry can bind ~300 μg of antibody, though this may vary for some antibodies. I usually incubate with an excess of (e.g. 350 μg) antibody. This is designed to saturate the beads with IgG, resulting in the most efficient IP possible. Beads can be generated with less antibody if necessary.

Solutions

Citrate Phosphate Buffer pH 5.00.2 M Triethanolamine (TEA) pH 8.2

25 mM Citric Acid3.71g Triethanolamine -HCl/ 100 mL

50 mM Sodium Phosphate (Na2HPO4)

1.) Wash the Protein G dynabeads with Citrate Phosphate buffer 3x

  • After adding the buffer, vortex the beads and place the tube in the magnetic rack.

It may be necessary to spin the tubes briefly (~1 second in mini-fuge) to get solution off of the cap. This is necessary because any beads in the cap won’t be attracted to the magnet. Keep this mind whenever you use the beads.

2.) For 1 mL slurry, prepare 350 μg of antibody. Bring the solution to 1mL with citrate phosphate buffer.

  • Take 10 μL of solution for analysis (sample 1)

3.) Remove the last wash from the beads and keep the tube on the magnet. Add the antibody solution. Rotate the tubes for at lease 1 hour at room temp.

  • After binding is complete, take 10 μL of supernatant for analysis (sample 2)

4.) After incubation, wash 3x with citrate phosphate, then 2x with 0.2M Triethanolamine (TEA) ph 8.2.

  • Take 20 μL of slurry for analysis (sample 3)

5.) Prepare a cross-linking solution of 20 mM Dimethyl Pimelimidate (DMP) in TEA (5.4mg DMP-2HCl/mL).

6.) Remove the last TEA wash from the beads using the magnet and add 1 mL of DMP solution. Incubate 30 minutes at room temp with rotation.

7.) Remove DMP and incubate beads with 50 mM Tris pH 7.5 for 15 minutes. This solution will soak up any leftover cross-linkers.

8.) Wash 3x w/ PBS 0.01% Tween 20 (PBS-T).

9.) Incubate the beads with 1 mL 0.1M Glycine pH 2.5-2.7 for 5 minutes (rotating @ 4oC). This will elute off un-crosslinked antibody.

  • Take 10 μL of Glycine wash for analysis (sample 4)

10.) Wash 3x more with PBS-T.

11.) Bring beads back to original packaged volume in PBS-T w/ .02% NaN3 for storage. For 1mL stock this takes 950 μL of solution. Store at 4oC until IP.

  • Take 20 μL of slurry for analysis (sample 5)

Assaying antibody binding

1.)To samples 1-5 collected above, add the appropriate volume of LSB (i.e. 1:1 2x LSB) and boil for 5 minutes.

2.)Run all the samples on a 12% SDS-PAGE followed by coomassie staining or western blotting with secondary alone to visualize the heavy and light chains of your IgG.

This will allow you to see if your antibody bound and if the antibody has been crosslinked to the beads. As indicated in the example below, successful binding is indicated a by a depletion of IgG between samples 1 and 2 (pre and post binding), and a strong IgG signal in sample 3 (beads after binding). Successful crosslinking is indicated by a lack of IgG elution following boiling of the beads in LSB (compare samples 3 and 5).

Figure 1:An example gel showing successful bead conjugation. Note the depletion of IgG after bead binding (compare lanes 1 and 2), the low amount of IgG eluting from beads following crosslinking (compare lanes 3 and 5).
Optimizing before Mass Spec

Prior to doing a large scale IP, it is important to optimize your immunoprecipitation conditions. This way, you will be confident in the quality of the samples you are producing. Here we will cover how to titrate beads to achieve an efficient IP, but there are a number of other parameters that can be optimized (e.g. lysis buffers, elution conditions, etc).

Titration of beads for immunoprecipitation

1.)Prepare a cell lysate, ideally under the same growth and lysis conditions you will use for your large-scale IP. A single 10 cm will provide ample material.

  1. Prepare 100 μL for SDS-PAGE

2.)Aliquot your target-antibody beads into several tubes, each with a different volume of beads. For example, a range of 2.5-100 μL of slurry.

3.)Using the magnet, wash the beads twice with lysis buffer.

4.)Remove all liquid from the beads and add 100 μL of lysate to each aliquot of beads

5.)Incubate lysate and beads for at least one hour at 4oC with rotation.

6.)Following incubation, save the supernatant for SDS-PAGE analysis.

7.)Wash the beads 3 times with lysis buffer

8.)Elute target protein by boiling beads in 2x LSB for 5 minutes (or using your preferred elution condition)

9.)Analyze samples by western blot analysis for your target protein plus a loading control (e.g. actin, tubulin, etc).

Analyze the original lysate (pre-IP) and each post-IP supernatant, along with each elution by western blot for your target protein. You should see depletion of your target protein in the post-IP supernatant with increasing volumes of beads (see example below). Keep in mind that complete depletion is not necessarily required for an efficient IP. In some cases where your target is very abundant, it may be difficult to see depletion, but analysis of your eluates will indicate the volume required for an efficient IP.

Figure 2: An example immuno-depletion experiment used to optimize p120 IP conditions. 100 μL lysate was incubated with the indicated volume of p120 or control bead slurry. Note the depletion of p120 following IP with increasing volumes of beads, but no depletion by control beads. Based on these results, 25 μL slurry/100 μL lysate (or 250 μL slurry/mL lysate) was determined to be the optimal bead volume for p120 IPs.

Large Scale Cell Lysis

Prior to cell lysis:

  • Prepare ice-water bath
  • Chill PBS
  • Prepare lysis buffer with protease and phosphatase inhibitors, keep lysis buffer cold
  • Pre-chill eppendorf tubes (1 2 mL tube for each 15 cm dish)

1.)Wash cells twice with PBS (10 mL/15 cm dish) to remove all traces of media.

2.)Place dishes on ice-water bath, and aspirate the last of the PBS from the dish.

3.)Add 1 mL of lysis buffer to each 15 cm dish; incubate cells on ice in lysis buffer for 5 minutes.

4.)Thoroughly scrape cells from the plate, pipette the lysate into a 2 mL eppendorf tube.

5.)Homogenize the cell lysate by passing the lysate through a needle and syringe 5 times.

6.)Centrifuge the lysate (in 2 mL eppendorf tubes) at 4oC for 10 minutes at maximum speed.

7.)Pool the clarified lysate into a single 15 mL conical tube.

  • Optional: estimate protein concentration with an appropriate assay (e.g. BCA)

Immunoprecipitation

Note: This protocol covers a standard immunoprecipitation for MS. For a detailed protocol of crosslinked IPs (ReCLIP), see the attached protocol.

Prior to Immunoprecipitation

  • Aliquot the appropriate volume of antibody beads and wash twice with lysis buffer
  • Prepare a whole cell lysate for SDS-PAGE analysis
  • Determine the appropriate volume of lysate for immunoprecipitation. Make sure you have sufficient material for at least 2 IPs: 1 for your target and 1 for your control IgG. The volumes and concentration should be identical for both.
  1. Using the magnet, remove the last of wash from the beads, place the tubes with beads on ice
  2. Add the appropriate volume of lysate to the beads.
  3. Rotate tubes 2-4 hours at 4oC
  4. Place IP tubes on the magnet, and save some supernatant for analysis, the rest can be discarded
  5. Wash beads at least 5 times with lysis buffer (supplemented with protease/phosphatase inhibitors).
  6. Remove all of the liquid from the final wash, proceed with elution
  7. There are a number of elution methods available, the ideal method should be selected before doing your large-scale IP. A few examples are below:
  • LSB: Boil the beads in 30 μL fresh 2x LSB for 5 minutes. This is the fastest, simplest, but also dirtiest method available.
  • Urea: Incubate beads with 20 μL 8.0 M urea buffer (50 mM Tris pH 8.5, 8 M Urea) for 10 minutes at room temperature with rotation. Repeat the elution as necessary.
  • Low pH: Incubate the beads in a low pH buffer (e.g. 0.1 M Glycine or 0.1 M citric acid, pH 2.5) for 5 minutes at room temperature, repeat elution as necessary.

Assessing your IP prior to Mass Spec

Before submitting your eluates for mass spec analysis, it is important to do some preliminary analysis to make sure your IP actually worked. Similar to the previous optimization steps, the best approach is to analyze the pre and post-IP lysates to detect immuno-depletion for your target, and to analyze a small fraction (1/10th) of your eluates to detect the successful IP and elution of your target.

Processing for Mass Spec

Once the IP is complete, samples must be processed for subsequent MS analysis. One of the most common approaches called “short-stack” (also called “Franklin Stack”) is described here. In principle, proteins are briefly separated by SDS-PAGE (i.e. a few centimeters into the gel) and the entire sample is excised and submitted for analysis.

Requirements

  • Fresh LSB
  • Pre-cast SDS-PAGE gels (for example Invitrogen’s Nu-PAGE gels)
  • Clean, unused razors or scalpels

1.)Boil eluates in fresh LSB (if not already in LSB) for 5 minutes.

2.)Load samples into a pre-cast SDS-PAGE gel

3.)Run SDS-PAGE gel briefly, samples should only run a few centimeters at the most before stopping the gel.

4.)Remove gel and proceed with coomassie staining.

5.)Following destain, excise the entire length of the sample, starting from the bottom of the well.

6.)Dice up the sample gel into smaller cubes, and place in a clean eppendorf tube.