Immunoprecipitation
A single-target immunoprecipitation: the antibody is used to concentrate one protein out of a lysate so it can be blotted, sequenced or assayed. The question is yield and purity, judged against an input lane and the unbound supernatant, and the usual failure is the antibody's own heavy chain landing on top of the target band.
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IP
A single-target immunoprecipitation: the antibody is used to concentrate one protein out of a lysate so it can be blotted, sequenced or assayed. The question is yield and purity, judged against an input lane and the unbound supernatant, and the usual failure is the antibody's own heavy chain landing on top of the target band.
How it differs from the standard method
- Yield is checked against a 2% input lane and against depletion of the supernatant, not by how dark the eluate band looks.
- Harsher buffers are usually better here: without a complex to preserve, stringency only costs background.
- An IP for a modification (phospho, ubiquitin, acetyl) needs the matching inhibitors in the lysis buffer or the modification is gone before capture.
- An IP destined for an activity assay or a structural method needs a native elution, which costs yield.
1One tube, two experiments
An immunoprecipitation uses an antibody to take one protein out of a complicated mixture. The antibody binds its target in the lysate, a resin carrying protein A or protein G (or a magnetic bead carrying an anti-tag antibody) collects the antibody, and everything that is not held on the beads is washed away. What you then run on a gel is an enormous enrichment of one protein, and of everything that came with it.
2The controls are the experiment
Two control tubes go through everything the real one does, from the same lysate. Beads with no antibody catch whatever binds the resin itself, and plenty does, because agarose, protein A/G and the blocking protein on the beads are all surfaces.
Pre-clear
Bare beads first, then discard them. Removes most of what binds the resin before the antibody is anywhere near it.
Capture
Antibody and lysate, rotating in the cold. Capture saturates with antibody; background does not.
Wash
Each wash removes background faster than signal, and gives a weakly held partner another chance to leave.
Elute
Boiling takes everything, including the antibody. Low pH or a competing peptide takes much less of it.
Blot
Input, unbound, bead-only, isotype and IP side by side, or the numbers mean nothing.
3Stringency: you cannot have both
The buffer decides what survives. A gentle 0.1% NP-40 buffer at physiological salt keeps a transient complex together, and it also keeps a great deal of non-specific binding, because detergent and salt are exactly what strip weak surface interactions.
4The antibody lands on your blot at 50 and 25 kDa
Whatever else is in the eluate, the antibody is, and reduced IgG runs as a heavy chain at about 50 kDa and a light chain at about 25 kDa. Those are two of the most crowded addresses on a protein gel.
- Pre-clearing
- Rotating the lysate with bare beads and throwing them away before adding antibody. Costs almost no target and removes most of the proteins that are attracted to the resin rather than to your antibody.
- Isotype control
- An irrelevant antibody of the same isotype, species and amount, taken through the identical procedure. The only lane that shows which bands bind immunoglobulin.
- Input and unbound
- A small fraction of the lysate before capture and of the supernatant after it. Together they say whether a weak band means poor capture or a protein that was never there.
- Native elution
- Releasing the complex without denaturing it: a competing peptide, a tag-specific elution, or low pH followed by immediate neutralisation. Essential if the eluate is going on to an activity assay.
- Crosslinking
- Fixing complexes with a reversible crosslinker in intact cells before lysis, so an interaction that would not survive the buffer still survives it. It also fixes everything that happened to be nearby.
5What a co-IP does and does not show
A co-IP shows that two proteins were in the same complex in a lysate, at the moment of lysis, under one buffer condition. It does not show that they touch: a bridging protein gives the same result, and the reciprocal experiment does not exclude one either.
Key terms
Practise these as flashcards →Bacterial proteins that bind the Fc region of IgG with different species and subclass preferences. Protein G binds mouse IgG1 well, where protein A is weak: a common reason an IP fails for no apparent reason.
How hard the buffer and washes are on non-covalent binding. Raising it removes background and complexes at the same time, in that order.
The 50 kDa and 25 kDa bands that reduced IgG puts on a gel. They swamp any target running nearby unless the elution or the detection keeps them off the blot.
A secondary antibody that binds native IgG and barely recognises reduced, denatured chains, so the antibody used for capture stays invisible on the blot.
The same beads and lysate with no antibody. Identifies everything that binds the resin, which is the largest single source of false partners.
How much of the target is gone from the supernatant after capture. The cheapest check that the antibody worked, and it needs nothing but a lane.
Pulling the candidate partner and blotting for the original bait. A different antibody and a different epitope, so it fails in different ways.
When to use it
- Finding or confirming an interaction partner of a protein in its own cellular context, with the complexes that exist in cells rather than in a tube.
- Enriching a low-abundance protein out of a lysate so it can be detected, sequenced, or assayed for a modification that a whole-lysate blot would never show.
- Testing whether an interaction responds to biology: a stimulus, a drug, a cell-cycle stage, or a point mutation in the predicted binding surface.
- Preparing a native complex for a downstream measurement: an activity assay, a mass-spectrometry identification, or an electron microscopy grid.
Compared with alternatives
Uses a recombinant bait on a resin instead of an antibody, so it can show direct binding between purified proteins, at the price of being outside the cell.
Tags neighbours inside living cells, so it sees interactions that do not survive lysis; prefer it when a co-IP keeps coming out empty.
Reports proximity in fixed cells with subcellular location preserved, which a lysate cannot; prefer it when where matters as much as whether.
Identifies everything in the eluate rather than the one protein you blotted for; the natural readout once the controls are trustworthy.
Measures affinity and kinetics for a purified pair; prefer it when the question is how tight and how fast, not whether the two are in a cell complex.
Screens for binary interactions without antibodies or lysates, and is a useful independent line of evidence for a candidate found by co-IP.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A single-target immunoprecipitation: the antibody is used to concentrate one protein out of a lysate so it can be blotted, sequenced or assayed. The question is yield and purity, judged against an input lane and the unbound supernatant, and the usual failure is the antibody's own heavy chain landing on top of the target band.
- Yield is checked against a 2% input lane and against depletion of the supernatant, not by how dark the eluate band looks.
- Harsher buffers are usually better here: without a complex to preserve, stringency only costs background.
- An IP for a modification (phospho, ubiquitin, acetyl) needs the matching inhibitors in the lysis buffer or the modification is gone before capture.
- An IP destined for an activity assay or a structural method needs a native elution, which costs yield.